HIN-1, a tumor suppressor gene

ABSTRACT

The invention encompasses isolated DNAs encoding HIN-1 polypeptides, vectors containing such DNAs, cells containing the vectors, and isolated HIN-1 polypeptides. The invention also features methods of making and using HIN-1 polypeptides.

[0001] This application claims priority, under 35 USC §119(e)(1), ofprior U.S. provisional application Nos. 60/270,973, filed Feb. 23, 2001,and 60/351,908, filed Jan. 25, 2002.

TECHNICAL FIELD

[0002] The invention relates to cancer, and more particularly to cancersuppressor genes.

BACKGROUND

[0003] Breast carcinoma is the second leading cause of cancer-relateddeath in women of the western world. In the United States alone over175,000 new cases are diagnosed annually. The natural history of breastcancer involves a sequential progression through defined clinical andpathologic stages starting with initially benign then atypicalhyperproliferation, progressing into in situ then invasive carcinomas,and culminating in metastatic disease. Ductal carcinoma in situ (DCIS)is the precursor of invasive ductal carcinoma.

SUMMARY

[0004] The invention is based on the identification of a human genethat, while highly expressed in normal breast tissue, is not expressedor is poorly expressed in DCIS tumors as well as breast tumors at otherstages. This gene has been designated the “HIN-1” (High in Normal 1)gene. The inventors have also identified homologues of the human HIN-1gene in mice and rats. Human HIN-1 is designated as hHIN-1, mouse HIN-1as mHIN-1, and rat HIN-1 as rHIN-1. Text that refers to HIN-1 withoutspecifying human, mouse or rat is pertinent to all three forms of HIN-1.The inventors have, in addition, identified two Drosophila genescontaining sequences encoding proteins with significant homology tohHIN-1. Thus, the invention features an isolated DNA encoding an HIN-1polypeptide, purified HIN-1 polypeptides, vectors containing the DNAs,and cells containing the vectors. In addition, the invention features amethod of making an HIN-1 polypeptide, in vitro and in vivo methods ofinhibiting proliferation of a cancer cell, and methods of diagnosingcancer.

[0005] More specifically, the invention features an isolated DNAcontaining a nucleic acid sequence encoding a polypeptide consisting ofSEQ ID NO:1 or SEQ ID NO:22. The DNA can, for example, include thenucleic acid sequence designated SEQ ID NO:3 or SEQ ID NO:23. Theinvention also includes a vector containing: (a) a nucleic acid sequencethat (i) encodes a polypeptide that inhibits proliferation of breastcancer cells, and (ii) hybridizes under highly stringent conditions to aprobe consisting of a sequence that is the complement of SEQ ID NO:3; or(b) the complement of the nucleic acid sequence. Vectors of theinvention can also contain any of the isolated DNAs of the invention. Inthe vectors, polypeptide encoding sequences can be operably linked to atranscriptional regulatory element (TRE). Also encompassed by theinvention is a cell (e.g., a prokaryotic or a eukaryotic cell)comprising any vector of the invention.

[0006] Also featured by the invention is an isolated polypeptidecontaining: (a) a protein that inhibits proliferation of breast cancercells and that is encoded by a nucleic acid sequence that hybridizesunder highly stringent conditions to a probe that includes or is thesequence that is the complement of SEQ ID NO:3; or (b) the protein,except for one or more conservative amino acid substitutions. Thepolypeptide can include the amino acid sequence of SEQ ID NO:1 or SEQ IDNO:22. Another polypeptide of the invention is an isolated polypeptidecontaining (a) a functional fragment of any of the above-describedpolypeptides; or (b) the functional fragment, except for one or moreconservative amino acid substitutions. Also included in the invention isa method of making a polypeptide; the method involves culturing a cellof the invention and extracting the polypeptide from the culture. Theinvention also features fragments of any of the DNAs of the invention,e.g., fragments of the DNA with SEQ ID NO:3 that include nucleotides 55and 56 of SEQ ID NO:3. The fragments of the DNAs of the invention willbe at least 10 bp, 15 bp, 25 bp, 50 bp, 75 bp, 100 bp, 125 bp, 150 bp,175 bp, 200 bp, 250 bp, 300 bp, 305 bp, or 309 bp long.

[0007] Another aspect of the invention is a method of inhibitingproliferation of a cancer cell. The method involves contacting thecancer cell with any of the polypeptides of the invention. The cancercell can be, for example, a breast cancer cell. The contacting can be invitro. Alternatively, the cancer cell can be in a mammal and thecontacting in the mammal can involve administering either thepolypeptide or a polynucleotide encoding the polypeptide to the mammal.Where the cancer cell is in a mammal, the method can involve: (a)providing a recombinant cell that is the progeny of a cell obtained fromthe mammal and has been transfected or transformed ex vivo with anucleic acid encoding the polypeptide; and (b) administering the cell tothe mammal.

[0008] Another embodiment of the invention is a method of identifying acompound that enhances inhibition of proliferation of cancer cells. Themethod involves: (a) providing a first and a second plurality of cancercells; (b) combining a test compound, the first plurality of cancercells, and any of the polypeptides of the invention; (c) combining thesecond plurality of cancer cells and; (d) determining the levelproliferation of the first plurality of cancer cells. A decreased levelof proliferation of the first plurality of cancer cells, as compared tothe second plurality of cells, indicates that the test compound enhancesinhibition of proliferation of cancer cells by the polypeptide.

[0009] Also featured by the invention is a method of diagnosis. Themethod can involve (a) providing a test cell; and b) measuring the levelof expression of a HIN-1 gene in the cell. Lack of expression of theHIN-1 gene or a low level of expression of the HIN-1 gene is anindication that the test cell is a cancer cell. Expression of the HIN-1gene can be measured as a function of the level of HIN-1 mRNA in thecell or as a function of the level of HIN-1 polypeptide in the cell.

[0010] In another aspect, the invention provides a method of diagnosis.The method involves (a) providing a test cell; and (b) determining thedegree of methylation of a HIN-1 promoter region in the test cell. Ahigh degree of methylation of the HIN-1 promoter region is an indicationthat the test cell is a cancer cell. The test cell can be, for example,a breast cell, a prostate cell, a pancreatic cell, or a lung cell.

[0011] The invention features an antibody that binds to any of thepolypeptides of invention. The antibody can be a monoclonal antibody ora polyclonal antibody.

[0012] Also included in the invention is a method of treatment thatinvolves identifying a patient as having cancer cells in which (a) HIN-1gene expression is low or (b) a HIN-1 promoter region is methylated; andtreating the patient with a compound that reduces methylation of theHIN-1 promoter region or with a compound that induces expression of agene with a methylated promoter region, e.g., the HIN-1 gene.

[0013] Yet another aspect of the invention is a method of identifying acompound that replaces the function of HIN-1 in cells that do notexpress HIN-1. The method involves: (a) providing a first cell that doesnot express HIN-1; (b) providing a second cell that does express HIN-1;(c) treating the first cell and the second cell with a test compound;and (d) determining whether the test compound decreases proliferation ofthe first or the second cell. A compound that decreases proliferation ofthe first cell but not the second cell can potentially replace thefunction of HIN-1 in cells that do not express HIN -1.

[0014] “Polypeptide” and “protein” are used interchangeably and mean anypeptide-linked chain of amino acids, regardless of length orpost-translational modification. The invention also features HIN-1polypeptides with conservative substitutions. Conservative substitutionstypically include substitutions within the following groups: glycine andalanine; valine, isoleucine, and leucine; aspartic acid and glutamicacid; asparagine, glutamine, serine and threonine; lysine, histidine andarginine; and phenylalanine and tyrosine.

[0015] As used herein, “full-length HIN-1” is HIN-1 with its nativesignal sequence.

[0016] The term “isolated” polypeptide or peptide fragment as usedherein refers to a polypeptide or a peptide fragment which either has nonaturally-occurring counterpart or has been separated or purified fromcomponents which naturally accompany it, e.g., in tissues such aspancreas, liver, spleen, ovary, testis, muscle, joint tissue, neuraltissue, gastrointestinal tissue or tumor tissue, or body fluids such asblood, serum, or urine. Typically, the polypeptide or peptide fragmentis considered “isolated” when it is at least 70%, by dry weight, freefrom the proteins and other naturally-occurring organic molecules withwhich it is naturally associated. Preferably, a preparation of apolypeptide (or peptide fragment thereof) of the invention is at least80%, more preferably at least 90%, and most preferably at least 99%, bydry weight, the polypeptide (or the peptide fragment thereof),respectively, of the invention. Thus, for example, a preparation ofpolypeptide x is at least 80%, more preferably at least 90%, and mostpreferably at least 99%, by dry weight, polypeptide x. Since apolypeptide that is chemically synthesized is, by its nature, separatedfrom the components that naturally accompany it, the syntheticpolypeptide is “isolated.”

[0017] An isolated polypeptide (or peptide fragment) of the inventioncan be obtained, for example, by extraction from a natural source (e.g.,from tissues or bodily fluids); by expression of a recombinant nucleicacid encoding the polypeptide; or by chemical synthesis. A polypeptidethat is produced in a cellular system different from the source fromwhich it naturally originates is “isolated,” because it will necessarilybe free of components which naturally accompany it. The degree ofisolation or purity can be measured by any appropriate method, e.g.,column chromatography, polyacrylamide gel electrophoresis, or HPLCanalysis.

[0018] An “isolated DNA” is either (1) a DNA that contains sequence notidentical to that of any naturally occurring sequence, or (2), in thecontext of a DNA with a naturally-occurring sequence (e.g., a cDNA orgenomic DNA), a DNA free of at least one of the genes that flank thegene containing the DNA of interest in the genome of the organism inwhich the gene containing the DNA of interest naturally occurs. The termtherefore includes a recombinant DNA incorporated into a vector, into anautonomously replicating plasmid or virus, or into the genomic DNA of aprokaryote or eukaryote. The term also includes a separate molecule suchas: a cDNA where the corresponding genomic DNA has introns and thereforea different sequence; a genomic fragment that lacks at least one of theflanking genes; a fragment of cDNA or genomic DNA produced by polymerasechain reaction (PCR) and that lacks at least one of the flanking genes;a restriction fragment that lacks at least one of the flanking genes; aDNA encoding a non-naturally occurring protein such as a fusion protein,mutein, or fragment of a given protein; and a nucleic acid which is adegenerate variant of a cDNA or a naturally occurring nucleic acid. Inaddition, it includes a recombinant nucleotide sequence that is part ofa hybrid gene, i.e., a gene encoding a non-naturally occurring fusionprotein. Also included is a recombinant DNA that includes a portion ofSEQ ID NO:3, SEQ ID NO:7, or SEQ ID NO:20. It will be apparent from theforegoing that isolated DNA does not mean a DNA present among hundredsto millions of other DNA molecules within, for example, cDNA or genomicDNA libraries or genomic DNA restriction digests in, for example, arestriction digest reaction mixture or an electrophoretic gel slice.

[0019] As used herein, a “functional fragment” of a HIN-1 polypeptide isa fragment of the polypeptide that is shorter than the full-lengthpolypeptide and has at least 5% (e.g., 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, 95%, 98%, 99%, 100%, or more) of the ability of thefull-length polypeptide to inhibit the proliferation of a cancer cell,e.g., a breast cancer cell. Fragments of interest can be made either byrecombinant, synthetic, or proteolytic digestive methods. Such fragmentscan then be isolated and tested for their ability to inhibit theproliferation of cancer cells as measured by [³H]-thymidineincorporation or cell counting.

[0020] As used herein, “operably linked” means incorporated into agenetic construct so that expression control sequences effectivelycontrol expression of a coding sequence of interest.

[0021] As used herein, the term “antibody” refers not only to wholeantibody molecules, but also to antigen-binding fragments, e.g., Fab,F(ab′)₂, Fv, and single chain Fv (ScFv) fragments. Also included arechimeric antibodies.

[0022] Unless otherwise defined, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention pertains. In case of conflict,the present document, including definitions, will control. Preferredmethods and materials are described below, although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention. All publications,patent applications, patents and other references mentioned herein areincorporated by reference in their entirety. The materials, methods, andexamples disclosed herein are illustrative only and not intended to belimiting.

[0023] Other features and advantages of the invention, e.g., inhibitingproliferation of cancer cells, will be apparent from the followingdescription, from the drawings and from the claims.

DESCRIPTION OF DRAWINGS

[0024]FIG. 1A is a depiction of the nucleotide sequence (SEQ ID NO:3) ofcDNA encoding the full-length hHIN-1 polypeptide.

[0025]FIG. 1B is a depiction of the nucleotide sequence (SEQ ID NO:4) ofcDNA encoding a mature hHIN-1 polypeptide (i.e., the full-length hHIN-1polypeptide but without an 18 amino acid signal peptide).

[0026]FIG. 1C is a depiction of the nucleotide sequence (SEQ ID NO:23)of cDNA encoding a mature hHIN-1 polypeptide (i.e., the full-lengthhHIN-1 polypeptide but without a 20 amino acid signal peptide).

[0027]FIG. 2A is a depiction of the amino acid sequence (SEQ ID NO:1) ofthe full-length hHIN-l polypeptide.

[0028]FIG. 2B is a depiction of the amino acid sequence (SEQ ID NO:2) ofa mature hHIN-1 polypeptide (i.e., the full-length hHIN-1 polypeptidebut without an 18 amino acid signal peptide).

[0029]FIG. 2C is a depiction of the amino acid sequence (SEQ ID NO:22)of a mature hHIN-1 polypeptide (i.e., the full length hHIN-1 polypeptidebut without a 20 amino acid signal peptide).

[0030]FIG. 3A is a depiction of the nucleotide sequence (SEQ ID NO:7) ofcDNA encoding the full-length mHIN-1 polypeptide.

[0031]FIG. 3B is a depiction of the nucleotide sequence (SEQ ID NO:8) ofcDNA encoding a mature mHIN-1 polypeptide (i.e., the full-length mHIN-1polypeptide but without a 19 amino acid signal peptide).

[0032]FIG. 3C is a depiction of the nucleotide sequence (SEQ ID NO:25)of cDNA encoding a mature mHIN-1 polypeptide (i.e., the full-lengthmHIN-1 polypeptide but without a 21 amino acid signal peptide).

[0033]FIG. 4A is a depiction of the amino acid sequence (SEQ ID NO:5) ofthe full-length mHIN-1 polypeptide.

[0034]FIG. 4B is a depiction of the amino acid sequence (SEQ ID NO:6) ofa mature mHIN-1 polypeptide (i.e., the full-length mHIN-1 polypeptidebut without a 19 amino acid signal peptide).

[0035]FIG. 4C is a depiction of the amino acid sequence (SEQ ID NO:24)of a mature mHIN-1 polypeptide (i.e., the full-length mHIN-1 polypeptidebut without a 21 amino acid signal peptide).

[0036]FIG. 5A is a photograph of an autoradiogram obtained from amultiple tissue mRNA expression array exposed to a ³²P-labeled hHIN-1cDNA probe (BR, breast; LU, lung; ES, esophagus; DU, duodenum; TR,trachea; PR, prostate; FL, fetal lung; FK, fetal kidney; PA, pancreas;LN, lymph nodes; NA, nucleus accumbens; and PI, pituitary gland). Thearrows indicate the position of a row of spots of mRNA from a range ofcancer lines including leukemias, lymphomas, lung cancer cells,colorectal cancer cells, and cervical cancer cells.

[0037]FIG. 5B is a photograph of an autoradiogram obtained from multipletissue northern blots exposed to a ³²P-labeled hHIN-1 cDNA probe.

[0038] FIGS. 5C-5E are photomicrographs showing in situ hybridization(dark staining) of a digitonin labeled hHIN-1 anti-sense ribo-probe tonormal mammary epithelium (20× magnification; FIG. 5C), normal mammaryepithelium (200× magnification; FIG. 5D) and DCIS mammary epithelium(200× magnification; FIG. 5E).

[0039]FIG. 5F is a photograph of an autoradiogram obtained from northernblots (exposed to a ³²-labeled hHIN-1 cDNA probe) of RNA isolated from:normal human mammary organoids (“organoids”) of three individualpatients; cultured primary human mammary epithelial cells (“HME”);mammary epithelial cells in a frozen section from a 25-week pregnantpatient (“Preg”); and five breast cancer cell lines.

[0040]FIG. 5G is a bar graph showing the results of a real-time PCRanalysis of hHIN-1 mRNA expression in laser capture microdissectedbreast cancer tissue.

[0041]FIG. 6A is a diagram showing the results of a sequence analysis ofbisulfite treated genomic DNA from the indicated cell lines. Each circlerepresents a potential methylation site and the intensity of circle-fillindicates the frequency at which the site was found to be methylated inthe PCR product clones analyzed. The darkest fill represents 100%, nofill represents 0% and three intermediate intensities represent 75%,50%, and 25%. Genomic DNA was extracted from ZR-75-1 cells (“ZR-75-1”),ZR-75-l cells cultured with 5 aza-cytosine (5aza-C) (“ZR-75-1-AC”),BT-549 cells (“BT-549”), SK-BR-3 cells (“SK-BR-3”) SUM159 cells(“SUM159”), SUM225 cells (“SUM225”), T44 cells (“T44”), normal mammaryepithelial cells from three separate patients (“Normal”), normal mammarycells from an 18-year old patient (“Normal (18 yo)”), normal mammarycells from a 34-year old patient (“Normal (34 yo)”), T47D cells(“T47D”), BT474 cells (“BT474”), ASCP cells (“ASCP”), PC3 cells (“PC3”),LNCP cells (“LNCP”), and pooled lung cancer tissue from four individualpatients (“Lung CA (4 samples)”), and analyzed. Also shown are relativelevels of hHIN-1 mRNA in relevant cells and tissues. “+++” indicateshigh levels of hHIN-1 mRNA that were detectable by Northern blotanalysis of 1 μg (or less) of total RNA (these levels were detected onlyin normal breast tissues). “+” indicates hHIN-1 mRNA levels detectableby Northern blot analysis of 5 g of total RNA. “-” indicates hHIN-1 mRNAlevels undetectable by Northern blot analysis. “+/−” for SUM225 cells isexplained in Example 4.

[0042]FIG. 6B is a photograph of an ethidium bromide stainedelecrophoretic gel of samples from an RT-PCR analysis of hHIN-1 mRNA inthe indicated breast cancer cell lines that were cultured in thepresence (“+”) and absence (“−”) of 5aza-C.

[0043]FIG. 6C is a photograph of an ethidium bromide stainedelecrophoretic gel of samples from a methylation-specific PCR analysisof the hHIN-1 proximal promoter region in genomic DNA from a series ofprimary breast tumors (upper panel) and breast cancer cell lines (lowerpanel). “M” and “U” indicate PCRs performed with methylated and theunmethylated sequence-specific primers, respectively.

[0044]FIG. 7 is a photograph of a Western blot. Cell lysates or culturemedia separated from cells were incubated with nickel-containing Ni-NTAbeads. Proteins bound to beads were eluted from the beads and subjectedto sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE) andthe SDS-PAGE gel was blotted onto a Western blot membrane which wasdeveloped with a rabbit polyclonal antibody specific for hHIN-1. Thecells and culture media tested were from cultures containing: (i) 293cells (first four lanes) transfected with pCEP4 (“C”), orpCEP4-Histag-HIN-1 constructs; or (ii) MCF10A or SUM159 cells infectedwith Ad-Track-GFP (“G”) or Ad-Track-Histag-HIN-1 (“H”) adenoviralvectors.

[0045]FIG. 8. is a depiction of the nucleotide sequence (SEQ ID NO:19)of a promoter region immediately 5′ of the coding sequence for thehHIN-1 polypeptide.

[0046]FIG. 9A is a depiction of the nucleotide sequence (SEQ ID NO:20)of 279 nucleotides of cDNA encoding 93 amino acid of the full-lengthrHIN-1 polypeptide.

[0047]FIG. 9B is a depiction of the amino acid sequence (SEQ ID NO:21)of 93 amino acids of the full-length rHIN-1 polypeptide.

[0048]FIG. 9C is a depiction of the nucleotide sequence (SE ID NO:26) ofcDNA encoding a mature rHIN-1 polypeptide (i.e., the full length rHIN-1polypeptide but without a signal peptide).

[0049]FIG. 9D is a depiction of the amino acid sequence (SEQ ID NO:27)of a mature rHIN-1 polypeptide (i.e., the full-length rHIN-1 polypeptidebut without a signal peptide).

[0050]FIG. 10 is a depiction of the amino acid sequences of hHIN-1(“Human HIN1”), (SEQ ID NO:1) mHIN-1 (“Mouse HINI”) (SEQ ID NO:5) and 93amino acids of rHIN-1 (“Rat HIN1”) (SEQ ID NO:21) aligned for maximalhomology. Amino acid residues common to more than one of the polypeptideare indicated at the relevant position under the sequences. Conservedresidues and identical residues are shaded. Possible N-terminal signalpeptides are indicated by a bold line and corresponding signal peptidecleavage sites are indicated by an arrow.

[0051]FIG. 11A is a photograph of a dot blot of a 76 human tissue RNAexpression array exposed to a human uteroglobin related protein-1(UGRP-1) cDNA detection probe.

[0052]FIGS. 11B and 11C are photographs of Northern blots of RNA from: avariety of adult mouse tissues (FIG. 11B); whole mouse embryos of avariety of embryonic ages (FIG. 11C); and a variety organs from mouseembryos at 18.5 days of embryonic life (“E 18.5”) (FIG. 11C) exposed tomouse mHIN-1, mouse UGRP-1, mouse C/EBPδ (“CEBP-δ”), and mouse β-actincDNA detection probes.

[0053]FIGS. 11D and 11E are photographs of ethidium bromide-stainedelectrophoretic gels of reverse transcriptase-PCR (RT-PCR) reactionsperformed to detect the presence of mHIN-1 mRNA, mouse UGRP-1 mRNA,mouse C/EBPδ mRNA, and mouse β-actin mRNA in RNA isolated from: mammaryglands of virgin female mice and female mice (lactating or involuting)at days 1, 4, 12, and 21 (D1, D4, D12, and D21, respectively) postpartum (FIG. 11D); and mammary glands from female mice at days 6.5, 8.5,10.5, 12.5, 14.5, 16.5, and 18.5 post coitum and mammary glands fromlactating female mice at day 1 post partum (“Lactating D1”) (FIG. 11E).Control PCR reactions using mouse lung cDNA as a template were alsoperformed (“Lung”) (FIGS. 1D and E). In FIG. 11D the “Involuting D21.A”and “Involuting D21.B” mammary glands were obtained from two independentmice at 21 days post partum.

[0054] FIGS. 12A-12O are photomicrographs of histological sections ofadult (FIGS. 12A-12C) and embryonic (FIGS. 12D-12L) mouse lung and theuterus of a pregnant mouse (FIGS. 2M-12O) exposed to digitonin-labeledmHIN-1 anti-sense and sense (control) RNA probes. Day of embryonic lifeand the use of the control sense probe are indicated in appropriatefigures. The sections shown in

[0055]FIGS. 12D, 12F, and 12G are transverse sections made at the levelof the trachea, the sections shown in

[0056]FIGS. 12E and 12J are transverse sections made at the level of thebronchi, and the section shown in

[0057]FIG. 12L is a transverse section made at the level of thebronchioli. Arrows indicate mHIN-1 mRNA expressing cells.Photomicrographs were taken using 2× (FIGS. 12D-12G, 12I, 12J, and 12L),10× (FIGS. 12A, 12B, 12H, 12K, and 12M-12O), and 20× (FIG. 2C and insetsof FIGS. 12L and 12N) objective lenses.

[0058]FIG. 13 is a photograph of an ethidium bromide-stainedelectrophoretic gel of RT-PCR reactions performed to detect the presencehHIN-1 (“HIN-1”) mRNA, mucin 2 (“MUC2”) mRNA, human squamous cell markertransglutaminase I (“TGase I”) mRNA, and human β-actin mRNA in RNAextracted from human primary bronchial epithelial cells cultured with(“+”) and without (“−”) all-trans-retinoic acid (“RA”) for 1, 2, 3, 5,7, and 9 days.

[0059]FIG. 14A is a depiction of the amino acid sequences of: humanUGRP-1 (SEQ ID NO:32), hHIN-1 (SEQ ID NO:1); the Drosophila GC130681protein (SEQ ID NO:28); and the Drosophila GC13674 protein (SEQ IDNO:30) aligned for optimal homology using MacVector3 software. Identicaland conserved amino acids are indicated with dark shading and lightshading, respectively.

[0060]FIG. 14B is a dendrogram generated using DNASTAR3 software and theJ. Hein algorithm indicating the phylogenetic relationship of a varietyof HIN-1 homologues.

[0061]FIG. 15 is a depiction of the nucleotide sequence of cDNA (SEQ IDNO:29) encoding the Drosophila GC130681 protein.

[0062]FIG. 16 is a depiction of the nucleotide sequence of cDNA (SEQ IDNO:31) encoding the Drosophila GC13674 protein.

DETAILED DESCRIPTION

[0063] Using the Serial Analysis of Gene Expression (SAGE) methodology,the inventors have identified a gene, the hHIN-1 gene, that is expressedin normal human mammary epithelial tissue, but either is not or isweakly expressed in the majority of breast carcinomas. Inspection of thenucleotide sequence of a cDNA clone that contained a region of 100%homology to the hHIN-1 tag identified by the SAGE methodology revealedthat the cDNA clone included a sequence (SEQ ID NO:3) encoding apolypeptide (the hHIN-1 polypeptide; SEQ ID NO:1) (FIG. 2A) of 104 aminoacid residues in length. Inspection of the hHIN-1 polypeptide's aminoacid sequence revealed the presence of two possible signal peptidecleavage sites, one after Ala₁₈ and another after Ala₂₀. Thus, thesignal peptide can be either 18 or 20 amino acids long. The amino acidsequence of the mature hHIN-1 polypeptide lacking the 18 amino acidsignal peptide is assigned SEQ ID NO:2 (FIG. 2B) and the nucleotidesequence of cDNA encoding this mature hHIN-1 polypeptide is assigned SEQID NO:4 (FIG. 1B). The amino acid sequence of the mature hHIN-1polypeptide lacking the 20 amino acid signal peptide is assigned SEQ IDNO:22 (FIG. 2C) and the nucleotide sequence of cDNA encoding this maturehHIN-1 polypeptide is assigned SEQ ID NO:23 (FIG. 1C). The presence of asignal peptide and a transfection analysis indicated that the hHIN-1polypeptide is a novel cytokine that inhibits the growth of at leastsome breast cancer cells. The inventors have also identified a mousecDNA homologue (mHIN-1) of hHIN-1 cDNA that encodes a polypeptide (themHIN-1 polypeptide) of the same length as hHIN-1. The full-length mHIN-1polypeptide is assigned SEQ ID NO:5 (FIG. 4A) and the cDNA encoding thefull-length mHIN-1 polypeptide is assigned SEQ ID NO:7 (FIG. 3A). Twopossible signal peptides were identified within the mHIN-1 full-lengthpolypeptide, one of 19 and 21 amino acids in length. The amino acidsequence of the mature mHIN-1 polypeptide lacking the 19 amino acidsignal peptide is assigned SEQ ID NO:6 (FIG. 4B) and the nucleotidesequence of cDNA encoding this mature mHIN-1 polypeptide is assigned SEQID NO:8 (FIG. 3B). The amino acid sequence of mature mHIN-1 polypeptidelacking the 21 amino acid signal is assigned SEQ ID NO:24 (FIG. 4C) andthe nucleotide sequence of cDNA encoding this mature mHIN-1 polypeptideis assigned SEQ ID NO:25 (FIG. 3C). In addition, the inventors havedefined a rat partial cDNA sequence (SEQ ID NO:20) (FIG. 9A) encoding alarge portion of the rat homologue (rHIN-1) (SEQ ID NO:21) (FIG. 9B) ofhHIN-1. A signal peptide cleavage site was identified. The amino acidsequence of the mature rHIN-1 polypeptide lacking the signal peptide isassigned SEQ ID NO:27 (FIG. 9D) and the nucleotide sequence of cDNAencoding this mature rHIN-1 polypeptide is assigned SEQ ID NO:26 (FIG.9C).

[0064] RNA hybridization studies showed that the hHIN-1 gene isexpressed highly in a number of other normal tissues, e.g., lung,trachea, salivary gland, prostate gland, esophagus, duodenum, fetallung, and fetal kidney. Lower expression of the hHIN-1 gene was seen inpancreas, pituitary gland, lymph node, and accumbens nucleus.Dramatically reduced expression of hHIN-1 RNA (compared to normal lungtissue) was seen in 40 primary lung tumors. Loss of hHIN-1 expression inbreast cancer was not due to mutational events, but rather, at least inthe majority of breast cancer cells in which lack of or decreased HIN-1gene expression was seen, was due to methylation of a CpG island in thehHIN-1 gene promoter region. Similarly, dramatically reduced hHIN-1expression was seen in a panel of human lung cancers and methylation ofthe hHIN-1 gene promoter region was observed in five out of nine lungtumors tested. Similar results were obtained with prostate andpancreatic cancer cells.

[0065] Developmental studies and the expression pattern in Drosophila oftwo proteins (protein GC130681 and protein GC13674) with low butsignificant amino acid homology to hHIN-1 indicate an evolutionarilyconserved role for HIN-1 in epithelial cell differentiation.

[0066] HIN-1 Nucleic Acid Molecules

[0067] The HIN-1 nucleic acid molecules of the invention can be cDNA,genomic DNA, synthetic DNA, or RNA, and can be double-stranded orsingle-stranded (i.e., either a sense or an antisense strand). Segmentsof these molecules are also considered within the scope of theinvention, and can be produced by, for example, the polymerase chainreaction (PCR) or generated by treatment with one or more restrictionendonucleases. A ribonucleic acid (RNA) molecule can be produced by invitro transcription. Preferably, the nucleic acid molecules encodepolypeptides that, regardless of length, are soluble under normalphysiological conditions.

[0068] The nucleic acid molecules of the invention can contain naturallyoccurring sequences, or sequences that differ from those that occurnaturally, but, due to the degeneracy of the genetic code, encode thesame polypeptide (for example, the polypeptides with SEQ ID NOS:1 and5). In addition, these nucleic acid molecules are not limited to codingsequences, e.g., they can include some or all of the non-codingsequences that lie upstream or downstream from a coding sequence.

[0069] The nucleic acid molecules of the invention can be synthesized(for example, by phosphoramidite-based synthesis) or obtained from abiological cell, such as the cell of a mammal. The nucleic acids can bethose of a human, non-human primate (e.g., monkey), mouse, rat, guineapig, cow, sheep, horse, pig, rabbit, dog, or cat. Combinations ormodifications of the nucleotides within these types of nucleic acids arealso encompassed.

[0070] In addition, the isolated nucleic acid molecules of the inventionencompass segments that are not found as such in the natural state.Thus, the invention encompasses recombinant nucleic acid molecules (forexample, isolated nucleic acid molecules encoding hHIN-1 or mHIN-1)incorporated into a vector (for example, a plasmid or viral vector) orinto the genome of a heterologous cell (or the genome of a homologouscell, at a position other than the natural chromosomal location).Recombinant nucleic acid molecules and uses therefor are discussedfurther below.

[0071] Techniques associated with detection or regulation of genes arewell known to skilled artisans. Such techniques can be used to diagnoseand/or treat disorders associated with aberrant HIN-1 expression.Nucleic acid molecules of the invention are discussed further below inthe context of their therapeutic utility.

[0072] A HIN-1 family gene or protein can be identified based on itssimilarity to the relevant HIN-1 gene or protein, respectively. Forexample, the identification can be based on sequence identity. Theinvention features isolated nucleic acid molecules which are at least50% (or 55%, 65%, 75%, 85%, 95%, or 98%) identical to: (a) a nucleicacid molecule that encodes the polypeptide of SEQ ID NOS: 1, 2, 5, 6,21, 22, 24, or 27; (b) the nucleotide sequence of SEQ ID NOS: 3, 4, 7,8, 20, 23, 25, or 26; and (c) a nucleic acid molecule which includes asegment of at least 30 (e.g., at least 40, 50, 60, 80, 100, 125, 150,175, 200, 250, 300, or 306) nucleotides of SEQ ID NO: 3, 7, or 20.

[0073] The determination of percent identity between two sequences isaccomplished using the mathematical algorithm of Karlin and Altschul,Proc. Natl. Acad. Sci. USA 90, 5873-5877, 1993. Such an algorithm isincorporated into the BLASTN and BLASTP programs of Altschul et al.(1990) J. Mol. Biol. 215, 403-410. BLAST nucleotide searches areperformed with the BLASTN program, score=100, wordlength=12, to obtainnucleotide sequences homologous to HIN-1-encoding nucleic acids. BLASTprotein searches are performed with the BLASTP program, score=50,wordlength=3, to obtain amino acid sequences homologous to the HIN-1polypeptide. To obtain gapped alignments for comparative purposes,Gapped BLAST is utilized as described in Altschul et al. (1997) NucleicAcids Res. 25, 3389-3402. When utilizing BLAST and Gapped BLASTprograms, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) are used (See http://www.ncbi.nlm.nih.gov).

[0074] Hybridization can also be used as a measure of homology betweentwo nucleic acid sequences. A HIN-1-encoding nucleic acid sequence, or aportion thereof, can be used as a hybridization probe according tostandard hybridization techniques. The hybridization of a HIN-1 probe toDNA or RNA from a test source (e.g., a mammalian cell) is an indicationof the presence of HIN-1 DNA or RNA in the test source. Hybridizationconditions are known to those skilled in the art and can be found inCurrent Protocols in Molecular Biology, John Wiley & Sons, N.Y.,6.3.1-6.3.6, 1991. Moderate hybridization conditions are defined asequivalent to hybridization in 2× sodium chloride/sodium citrate (SSC)at 30° C., followed by a wash in 1× SSC, 0.1% SDS at 50° C. Highlystringent conditions are defined as equivalent to hybridization in 6×sodium chloride/sodium citrate (SSC) at 45° C., followed by a wash in0.2× SSC, 0.1% SDS at 65° C.

[0075] The invention also encompasses: (a) vectors (see below) thatcontain any of the foregoing HIN-1-related coding sequences and/or theircomplements (that is, “antisense” sequences); (b) expression vectorsthat contain any of the foregoing HIN-1-related coding sequencesoperably linked to any transcriptional/translational regulatory elements(examples of which are given below) necessary to direct expression ofthe coding sequences; (c) expression vectors encoding, in addition to aHIN-1 polypeptide, a sequence unrelated to HIN-1, such as a reporter, amarker, or a signal peptide fused to HIN-1; and (d) geneticallyengineered host cells (see below) that contain any of the foregoingexpression vectors and thereby express the nucleic acid molecules of theinvention.

[0076] Recombinant nucleic acid molecules can contain a sequenceencoding HIN-1 or HIN-1 having an heterologous signal sequence. The fulllength HIN-1 polypeptide, or a fragment thereof, may be fused to suchheterologous signal sequences or to additional polypeptides, asdescribed below. Similarly, the nucleic acid molecules of the inventioncan encode the mature form of HIN-1 or a form that includes an exogenouspolypeptide that facilitates secretion.

[0077] The transcriptional/translational regulatory elements referred toabove and further described below include but are not limited toinducible and non-inducible promoters, enhancers, operators and otherelements that are known to those skilled in the art and that drive orotherwise regulate gene expression. Such regulatory elements include butare not limited to the cytomegalovirus hCMV immediate early gene, theearly or late promoters of SV40 adenovirus, the lac system, the trpsystem, the TAC system, the TRC system, the major operator and promoterregions of phage A, the control regions of fd coat protein, the promoterfor 3-phosphoglycerate kinase, the promoters of acid phosphatase, andthe promoters of the yeast α-mating factors.

[0078] Similarly, the nucleic acid can form part of a hybrid geneencoding additional polypeptide sequences, for example, a sequence thatfunctions as a marker or reporter. Examples of marker and reporter genesinclude β-lactamase, chloramphenicol acetyltransferase (CAT), adenosinedeaminase (ADA), aminoglycoside phosphotransferase (neo^(r), G418^(r)),dihydrofolate reductase (DHFR), hygromycin-B-phosphotransferase (HPH),thymidine kinase (TK), lacZ (encoding β-galactosidase), and xanthineguanine phosphoribosyltransferase (XGPRT). As with many of the standardprocedures associated with the practice of the invention, skilledartisans will be aware of additional useful reagents, for example,additional sequences that can serve the function of a marker orreporter. Generally, the hybrid polypeptide will include a first portionand a second portion; the first portion being a HIN-1 polypeptide andthe second portion being, for example, the reporter described above oran Ig constant region or part of an Ig constant region, e.g., the CH2and CH3 domains of IgG2a heavy chain. Other hybrids could include anantigenic tag or His tag to facilitate purification.

[0079] The expression systems that may be used for purposes of theinvention include but are not limited to microorganisms such as bacteria(for example, E. coli and B. subtilis) transformed with recombinantbacteriophage DNA, plasmid DNA, or cosmid DNA expression vectorscontaining the nucleic acid molecules of the invention; yeast (forexample, Saccharomyces and Pichia) transformed with recombinant yeastexpression vectors containing the nucleic acid molecule of theinvention; insect cell systems infected with recombinant virusexpression vectors (for example, baculovirus) containing the nucleicacid molecule of the invention; plant cell systems infected withrecombinant virus expression vectors (for example, cauliflower mosaicvirus (CaMV) or tobacco mosaic virus (TMV)) or transformed withrecombinant plasmid expression vectors (for example, Ti plasmid)containing a HIN-1 nucleotide sequence; or mammalian cell systems (forexample, COS, CHO, BHK, 293, VERO, HeLa, MDCK, WI38, and NIH 3T3 cells)harboring recombinant expression constructs containing promoters derivedfrom the genome of mammalian cells (for example, the metallothioneinpromoter) or from mammalian viruses (for example, the adenovirus latepromoter and the vaccinia virus 7.5K promoter). Also useful as hostcells are primary or secondary cells obtained directly from a mammal andtransfected with a plasmid vector or infected with a viral vector.

[0080] Polypeptides and Polypeptide Fragments

[0081] The polypeptides of the invention include all those disclosedherein. They can be, for example, hHIN-1 (SEQ ID NO:1), hHIN-1 without asignal peptide (SEQ ID NO:2 or SEQ ID NO:22), mHIN-1 (SEQ ID NO:5),mHIN-1 without a signal peptide (SEQ ID NO:6 or SEQ ID NO:24), most ofrHIN-1 (SEQ ID NO:21), rHIN-1 without a signal peptide (SEQ ID NO:27)and functional fragments of these polypeptides. The polypeptidesembraced by the invention also include fusion proteins that containeither full-length HIN-1 or a functional fragment of it fused tounrelated amino acid sequence. The unrelated sequences can be additionalfunctional domains or signal peptides. Signal peptides are described ingreater detail and exemplified below. The polypeptides can be any ofthose described above but with one or more (e.g., one, two, three, four,five, six, seven, eight, nine, 10, 12, 14, 17, 20, 25, 30, 35, 40, 50,60, 70, 80, 90, 100 or more) conservative substitutions.

[0082] The polypeptides can be purified from natural sources (e.g.,blood, serum, plasma, tissues or cells such as normal breast epithelialcells or any cell that naturally produces HIN-1 polypeptides). Smallerpeptides (less than 50 amino acids long) can also be convenientlysynthesized by standard chemical means. In addition, both polypeptidesand peptides can be produced by standard in vitro recombinant DNAtechniques and in vivo transgenesis, using nucleotide sequences encodingthe appropriate polypeptides or peptides. Methods well-known to thoseskilled in the art can be used to construct expression vectorscontaining relevant coding sequences and appropriatetranscriptional/translational control signals. See, for example, thetechniques described in Sambrook et al., Molecular Cloning: A LaboratoryManual (2nd Ed.) [Cold Spring Harbor Laboratory, N.Y., 1989], andAusubel et al., Current Protocols in Molecular Biology [Green PublishingAssociates and Wiley Interscience, N.Y., 1989].

[0083] Polypeptides and fragments of the invention also include thosedescribed above, but modified for in vivo use by the addition, at theamino- and/or carboxyl-terminal ends, of a blocking agent to facilitatesurvival of the relevant polypeptide in vivo. This can be useful inthose situations in which the peptide termini tend to be degraded byproteases prior to cellular uptake. Such blocking agents can include,without limitation, additional related or unrelated peptide sequencesthat can be attached to the amino and/or carboxyl terminal residues ofthe peptide to be administered. This can be done either chemicallyduring the synthesis of the peptide or by recombinant DNA technology bymethods familiar to artisans of average skill.

[0084] Alternatively, blocking agents such as pyroglutamic acid or othermolecules known in the art can be attached to the amino and/or carboxylterminal residues, or the amino group at the amino terminus or carboxylgroup at the carboxyl terminus can be replaced with a different moiety.Likewise, the peptides can be covalently or noncovalently coupled topharmaceutically acceptable “carrier” proteins prior to administration.

[0085] Also of interest are peptidomimetic compounds that are designedbased upon the amino acid sequences of the functional peptide fragments.Peptidomimetic compounds are synthetic compounds having athree-dimensional conformation (i.e., a “peptide motif”) that issubstantially the same as the three-dimensional conformation of aselected peptide. The peptide motif provides the peptidomimetic compoundwith the ability to inhibit the proliferation of cancer cells (e.g.,breast cancer cells) in a manner qualitatively identical to that of theHIN-1 functional fragment from which the peptidomimetic was derived.Peptidomimetic compounds can have additional characteristics thatenhance their therapeutic utility, such as increased cell permeabilityand prolonged biological half-life.

[0086] The peptidomimetics typically have a backbone that is partiallyor completely non-peptide, but with side groups that are identical tothe side groups of the amino acid residues that occur in the peptide onwhich the peptidomimetic is based. Several types of chemical bonds,e.g., ester, thioester, thioamide, retroamide, reduced carbonyl,dimethylene and ketomethylene bonds, are known in the art to begenerally useful substitutes for peptide bonds in the construction ofprotease-resistant peptidomimetics.

[0087] Methods of Inhibiting Proliferation of a Cancer Cell

[0088] The methods of the invention involve contacting a cancer cellwith a HIN-1 polypeptide of the invention, or a functional fragmentthereof, in order to inhibit proliferation of the cancer cell. Suchpolypeptides or functional fragments can have amino acid sequencesidentical to wild-type sequences or they can contain one or more (e.g.,two, three, four, five, six, seven, eight, nine, 10, 12, 14, 17, 20, 25,30, 35, 40, 50, 60, 70, 80, 90, 100 or more) conservative amino acidsubstitutions. Cancer cells can be breast cancer, lung cancer, coloncancer, pancreatic cancer, renal cancer, stomach cancer, liver cancer,bone cancer, hematological cancer (e.g., leukemia or lymphoma), neuraltissue cancer, melanoma, ovarian cancer, testicular cancer, prostatecancer, cervical cancer, vaginal cancer, or bladder cancer cells.

[0089] The methods can be performed in vitro, in vivo, or ex vivo. Invitro application of HIN-1 can be useful, for example, in basicscientific studies of tumor cell biology, e.g., studies on signaltransduction or cell cycle analysis. In addition, HIN-1 and thepolynucleotides of the invention (DNA and/or RNA) can be used as“positive controls” in diagnostic assays (see below). However, themethods of the invention will preferably be in vivo or ex vivo (seebelow).

[0090] The HIN-1 proteins and variants thereof are generally useful ascancer cell (e.g., breast cancer cell) proliferation-inhibitingtherapeutics. They can be administered to mammalian subjects (e.g.,human breast cancer patients) alone or in conjunction with such drugsand/or radiotherapy.

[0091] These methods of the invention can be applied to a wide range ofspecies, e.g., humans, non-human primates, horses, cattle, pigs, sheep,goats, dogs, cats, rabbits, guinea pigs, hamsters, rats, and mice.

[0092] In vivo Approaches

[0093] In one in vivo approach, the HIN-1 polypeptide (or a functionalfragment thereof) itself is administered to the subject. Generally, thecompounds of the invention will be suspended in apharmaceutically-acceptable carrier (e.g., physiological saline) andadministered orally or by intravenous infusion, or injectedsubcutaneously, intramuscularly, intrathecally, intraperitoneally,intrarectally, intravaginally, intranasally, intragastrically,intratracheally, or intrapulmonarily. They are preferably delivereddirectly to tumor cells, e.g., to a tumor or a tumor bed followingsurgical excision of the tumor, in order to kill any remaining tumorcells. The dosage required depends on the choice of the route ofadministration; the nature of the formulation; the nature of thepatient's illness; the subject's size, weight, surface area, age, andsex; other drugs being administered; and the judgment of the attendingphysician. Suitable dosages are in the range of 0.01-100.0 μg/kg. Widevariations in the needed dosage are to be expected in view of thevariety of polypeptides and fragments available and the differingefficiencies of various routes of administration. For example, oraladministration would be expected to require higher dosages thanadministration by i.v. injection. Variations in these dosage levels canbe adjusted using standard empirical routines for optimization as iswell understood in the art. Administrations can be single or multiple(e.g., 2-, 3-, 4-, 6-, 8-, 10-, 20-, 50-,100-, 150-, or more fold).Encapsulation of the polypeptide in a suitable delivery vehicle (e.g.,polymeric microparticles or implantable devices) may increase theefficiency of delivery, particularly for oral delivery.

[0094] Alternatively, a polynucleotide containing a nucleic acidsequence encoding a HIN-1 polypeptide or functional fragment can bedelivered to cancer cells in a mammal. Expression of the coding sequencewill preferably be directed to lymphoid tissue of the subject by, forexample, delivery of the polynucleotide to the lymphoid tissue.Expression of the coding sequence can be directed to any cell in thebody of the subject. However, expression will preferably be directed tocells in the vicinity of the tumor cells whose proliferation it isdesired to inhibit. In certain embodiments, expression of the codingsequence can be directed to the tumor cells themselves. This can beachieved by, for example, the use of polymeric, biodegradablemicroparticle or microcapsule delivery devices known in the art.

[0095] Another way to achieve uptake of the nucleic acid is usingliposomes, prepared by standard methods. The vectors can be incorporatedalone into these delivery vehicles or co-incorporated withtissue-specific or tumor-specific antibodies. Alternatively, one canprepare a molecular conjugate composed of a plasmid or other vectorattached to poly-L-lysine by electrostatic or covalent forces.Poly-L-lysine binds to a ligand that can bind to a receptor on targetcells [Cristiano et al. (1995), J. Mol. Med. 73, 479]. Alternatively,tissue specific targeting can be achieved by the use of tissue-specifictranscriptional regulatory elements (TRE) which are known in the art.Delivery of “naked DNA” (i.e., without a delivery vehicle) to anintramuscular, intradermal, or subcutaneous site is another means toachieve in vivo expression.

[0096] In the relevant polynucleotides (e.g., expression vectors), thenucleic acid sequence encoding the HIN-1 polypeptide or functionalfragment of interest with an initiator methionine and optionally atargeting sequence is operatively linked to a promoter orenhancer-promoter combination.

[0097] Short amino acid sequences can act as signals to direct proteinsto specific intracellular compartments. Such signal sequences aredescribed in detail in U.S. Pat. No. 5,827,516, incorporated herein byreference in its entirety.

[0098] Enhancers provide expression specificity in terms of time,location, and level. Unlike a promoter, an enhancer can function whenlocated at variable distances from the transcription initiation site,provided a promoter is present. An enhancer can also be locateddownstream of the transcription initiation site. To bring a codingsequence under the control of a promoter, it is necessary to positionthe translation initiation site of the translational reading frame ofthe peptide or polypeptide between one and about fifty nucleotidesdownstream (3′) of the promoter. The coding sequence of the expressionvector is operatively linked to a transcription terminating region.

[0099] Suitable expression vectors include plasmids and viral vectorssuch as herpes viruses, retroviruses, vaccinia viruses, attenuatedvaccinia viruses, canary pox viruses, adenoviruses and adeno-associatedviruses, among others.

[0100] Polynucleotides can be administered in a pharmaceuticallyacceptable carrier. Pharmaceutically acceptable carriers arebiologically compatible vehicles that are suitable for administration toa human, e.g., physiological saline or liposomes. A therapeuticallyeffective amount is an amount of the polynucleotide that is capable ofproducing a medically desirable result (e.g., decreased proliferation ofcancer cells) in a treated animal. As is well known in the medical arts,the dosage for any one patient depends upon many factors, including thepatient's size, body surface area, age, the particular compound to beadministered, sex, time and route of administration, general health, andother drugs being administered concurrently. Dosages will vary, but apreferred dosage for administration of polynucleotide is fromapproximately 10⁶ to 10¹² copies of the polynucleotide molecule. Thisdose can be repeatedly administered, as needed. Routes of administrationcan be any of those listed above.

[0101] One alternative in vivo approach involves administering to asubject (e.g., a breast or lung tumor patient) having cancer cells inwhich the HIN-1 gene is low and/or a HIN-1 promoter region is methylateda compound that reduces methylation of the HIN-1 promoter region. Onesuch compound is 5-aza-2′-deoxycytidine. Another approach involvesadministration of histone deacetylase inhibitors (e.g., trichostatin orsodium butyrate) which induce expression of methylated genes. Suchcompounds could induce expression of HIN-1 in cells (e.g., breast cancercells) that either express it poorly or do not express it at all. Doses,frequency of doses, and routes of administration of methylation andhistone deacetylase inhibitors will be as described above for HIN-1polypeptides and functional fragments thereof. Human patients can betreated by, for example, one or more (e.g., two, three, four, five, six,seven eight, nine, or ten) intravenous infusions of5-aza-2′-deoxycytidine (100-1,000 mg/m²)

[0102] Ex vivo Approaches

[0103] An ex vivo strategy can involve transfecting or transducing cellsobtained from the subject with a polynucleotide encoding an HIN-1polypeptide or functional fragment-encoding nucleic acid sequencesdescribed above. The transfected or transduced cells are then returnedto the subject. The cells can be any of a wide range of types including,without limitation, hemopoietic cells (e.g., bone marrow cells,macrophages, monocytes, dendritic cells, T cells, or B cells),fibroblasts, epithelial cells, endothelial cells, keratinocytes, ormuscle cells. Such cells act as a source of the HIN-1 polypeptide orfunctional fragment for as long as they survive in the subject.Alternatively, tumor cells, preferably obtained from the subject butpotentially from an individual other than the subject, can betransfected or transformed by a vector encoding a HIN-1 polypeptide orfunctional fragment thereof. The tumor cells, preferably treated with anagent (e.g., ionizing irradiation) that ablates their proliferativecapacity, are then introduced into the patient, where they secreteexogenous HIN-1.

[0104] The ex vivo methods include the steps of harvesting cells from asubject, culturing the cells, transducing them with an expressionvector, and maintaining the cells under conditions suitable forexpression of the HIN-1 polypeptide or functional fragment. Thesemethods are known in the art of molecular biology. The transduction stepis accomplished by any standard means used for ex vivo gene therapy,including calcium phosphate, lipofection, electroporation, viralinfection, and biolistic gene transfer. Alternatively, liposomes orpolymeric microparticles can be used. Cells that have been successfullytransduced can then be selected, for example, for expression of thecoding sequence or of a drug resistance gene. The cells may then belethally irradiated (if desired) and injected or implanted into thepatient.

[0105] Methods of Screening for Compounds that Enhance the Ability ofHIN-1 to Inhibit Proliferation of Cancer Cells.

[0106] The invention provides methods for identifying compounds (smallmolecules or macromolecules) that enhance the ability of HIN-1 toinhibit proliferation of cancer cells. Such a method can involve, e.g.,culturing a HIN-1 polypeptide of the invention (or a functional fragmentthereof) with cancer cells in the presence of a test compound. Cancercells can be any of those disclosed herein. Useful HIN-1 polypeptidesinclude those with amino acid sequences identical to wild-type sequencesor they can contain one or more (e.g., one, two, three, four, five, six,seven, eight, nine, 10, 12, 14, 17, 20, 25, 30, 35, 40, 50, 60, 70, 80,90, 100 or more) conservative substitutions. The HIN-1 polypeptide canbe natural or recombinant. Compounds that enhance the inhibition by theHIN-1 polypeptide of proliferation of the cancer cells will likely becompounds that inhibit tumor growth.

[0107] A candidate compound whose presence requires at least 1.5 fold(e.g., 2-fold, 4-fold, 6-fold, 10-fold, 100-fold, 1000-fold, 10,000fold, or 100,000-fold) less HIN-1 to achieve a defined arbitrary levelof inhibition of cancer cell proliferation than achieved in the absenceof the compound can be useful for enhancing inhibition of cancer cellproliferation, and thus can be useful as a cancer therapeutic agent.

[0108] The invention also relates to using HIN-1 or functional fragmentsthereof to screen for compounds that can interact with HIN-1 andpotentially thereby enhance its ability to inhibit the proliferation ofcancer cells. One of skill in the art would know how to use standardmolecular modeling or other techniques to identify small molecules thatwould bind to appropriate sites (e.g., allosteric sites) on HIN-1. Onesuch example is provided in Broughton (1997) Curr. Opin. Chem. Biol. 1,392-398.

[0109] The invention also features a method of identifying a compound(small molecule or macromolecule) that can replace the function of HIN-1in a cell that does not express HIN-1. The method involves exposing both(1) cells not expressing HIN-1 and (2) cells expressing HIN-1 to a testcompound and determining whether the compound inhibits (i.e., totallyabrogates or diminishes in part) the proliferation of either type ofcells. Any compound that decreases the proliferation of cells notexpressing HIN-1 but either does not inhibit the proliferation of cellsexpressing HIN-1 or inhibits the proliferation of cells expressing HIN-1to a lesser degree (e.g., 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%,97%, 98%, or 99% less) than it does the proliferation of cells notexpressing HIN-1, is potentially a compound that replaces the functionof HIN-1 in cells not expressing HIN-1. The cells expressing the HIN-1can be any of the cells disclosed herein as expressing HIN-1 (e.g.,normal breast epithelial cells or certain breast cancer cells). Thecells can express HIN-1 from endogenous HIN-1 genes or they can berecombinant cells expressing, for example, a gene either stably ortransiently transfected into the cells. The cells not expressing HIN-1can be any cells that naturally (e.g., because of mutation or endogenousregulatory mechanisms) lack expression of HIN-1, e.g., many breastcancer cell lines or any of a variety of normal or tumor cells disclosedherein. The cells can also be those in which lack of HIN-1 expression isartificially induced, e.g., by gene “knockout” technology, antisensemethodologies, ribozyme, or RNAi methodologies. All these techniques arefamiliar to those in the art. The cells lacking expression of HIN-1 andthose expressing HIN-1 will preferably be of the same histological type.Treatment of the cells with the test compound can be carried out byculturing the cells with the test compound and measuring their level ofproliferation. Alternatively, the cells can be exposed to the testcompound for a period of time (e.g., one minute, 10 minutes, 30 minutes,one hour, two hours, four hours, eight hours, 12 hours, 18 hours, 24hours, two days, three days, 1 week, two weeks, 1 month, 2 months, threemonths or longer), after which the test compound is removed, and thecells are cultured for an additional period of time (e.g., one minute,10 minutes, 30 minutes, one hour, two hours, four hours, eight hours, 12hours, 18 hours, 24 hours, two days, three days, 1 week, two weeks, 1month, 2 months, three months or longer) and their proliferation ismeasured. Methods of determining relative levels of cell proliferationare known in the art, e.g., measurement of [³H]-thymidine incorporationinto the DNA of the cells or cell counting using, optionally, a vitalstain or a dye that is excluded by viable cells, e.g., trypan blue oreosin.

[0110] HIN-1 Antibodies

[0111] The invention features antibodies that bind to either or both ofthe HIN-1 polypeptides or fragments of such polypeptides. Suchantibodies can be polyclonal antibodies present in the serum or plasmaof animals (e.g., mice, rabbits, rats, guinea pigs, sheep, horses,goats, cows, or pigs) which have been immunized with the relevant HIN-1polypeptide or peptide fragment using methods, and optionally adjuvants,known in the art. Such polyclonal antibodies can be isolated from serumor plasma by methods known in the art. Monoclonal antibodies that bindto the above polypeptides or fragments are also encompassed by theinvention. Methods of making and screening monoclonal antibodies arewell known in the art.

[0112] Once the desired antibody-producing hybridoma has been selectedand cloned, the resultant antibody can be produced by a number ofmethods known in the art. For example, the hybridoma can be cultured invitro in a suitable medium for a suitable length of time, followed bythe recovery of the desired antibody from the supernatant. The length oftime and medium are known or can be readily determined.

[0113] Additionally, recombinant antibodies specific for HIN-1, such aschimeric and humanized monoclonal antibodies comprising both human andnon-human portions, are within the scope of the invention. Such chimericand humanized monoclonal antibodies can be produced by recombinant DNAtechniques known in the art, for example, using methods described inRobinson et al., International Patent Publication PCT/US86/02269; Akiraet al., European Patent Application 184,187; Taniguchi, European PatentApplication 171,496; Morrison et al., European Patent Application173,494; Neuberger et al., PCT Application WO 86/01533; Cabilly et al.,U.S. Pat. No. 4,816,567; Cabilly et al., European Patent Application125,023; Better et al. (1988) Science 240, 1041-43; Liu et al. (1987) J.Immunol. 139, 3521-26; Sun et al. (1987) PNAS 84, 214-18; Nishimura etal. (1987) Canc. Res. 47, 999-1005; Wood et al. (1985) Nature 314,446-49; Shaw et al. (1988) J. Natl. Cancer Inst. 80, 1553-59; Morrison,(1985) Science 229, 1202-07; Oi et al. (1986) BioTechniques 4, 214;Winter, U.S. Pat. No. 5,225,539; Jones et al. (1986) Nature 321, 552-25;Veroeyan et al. (1988) Science 239, 1534; and Beidler et al. (1988) J.Immunol. 141, 4053-60.

[0114] Also included within the scope of the invention are antibodyfragments and derivatives which contain at least the functional portionof the antigen binding domain of an antibody that binds specifically toHIN-1. Antibody fragments that contain the binding domain of themolecule can be generated by known techniques. For example, suchfragments include, but are not limited to: F(ab′)2 fragments which canbe produced by pepsin digestion of antibody molecules; Fab fragmentswhich can be generated by reducing the disulfide bridges of F(ab′)₂fragments; and Fab fragments which can be generated by treating antibodymolecules with papain and a reducing agent. See, e.g., NationalInstitutes of Health, 1 Current Protocols In Immunology, Coligan et al.,ed. 2.8, 2.10 (Wiley Interscience, 1991). Antibody fragments alsoinclude Fv (e.g., single chain Fv (scFv)) fragments, i.e., antibodyproducts in which there are few or no constant region amino acidresidues. An ScFv fragment is a single polypeptide chain that includesboth the heavy and light chain variable regions of the antibody fromwhich the ScFv is derived. Such fragments can be produced, for example,as described in U.S. Pat. No. 4,642,334, which is incorporated herein byreference in its entirety.

[0115] Methods of Diagnosis

[0116] The invention also features diagnostic assays. Such assays arebased on the findings that: (a) the hHIN-1 gene is either not expressedor is poorly expressed in a majority of breast and lung cancer cellswhile it is highly expressed in normal breast and lung tumors; and (b)many or most C residues in CpG sequences in a CpG island of the 5′promoter region of the hHIN-1 gene are methylated in the majority ofbreast tumors while none or very few of such residues are methylated innormal breast tissue. Thus, findings of either (a) no or low expressionof the HIN-1 gene in test cells; or (b) methylation of many or most Cresidues in CpG sequences in a CpG island of the 5′ promoter region ofthe hHIN-1 gene in test cells, would indicate that the test cells arecancer cells. Such tests can be used on their own or, preferably, inconjunction with other procedures to test for cancer in appropriatesubjects, e.g., human breast cancer patients. Test cells can be anycells with the potential to become or be any of the cancer cells listedherein. Thus, they can be, for example, breast cells, lung cells,prostate cells, pancreatic cells, gastrointestinal (e.g., colon) cells,or skin cells (e.g., melanocytes).

[0117] The level of expression of HIN-1 genes in test cells can measuredby any of a variety of methods known in the art. In general, suchmethods measure the level of either HIN-1 mRNA or HIN-1 polypeptide intest cells. In order to measure mRNA levels, test cells can be lysed andthe levels of HIN-1 mRNA in the lysates or in RNA purified orsemi-purified from the lysates determined by any of a variety of methodsfamiliar to those in the art. Such methods include, without limitation,hybridization assays using detectably labeled HIN-1 specific DNA or RNAprobes (see Example 3) and quantitative or semi-quantitative RT-PCRmethodologies using appropriate HIN-1 gene-specific oligonucleotideprimers. Alternatively, quantitative or semi-quantitative in situhybridization assays can be carried out using, for example, tissuesections or unlysed cell suspensions, and detectably (e.g.,fluorescently or enzyme) labeled DNA or RNA probes. Additional methodsfor quantitating mRNA include the RNA protection assay (RPA) and SAGE.

[0118] Methods of measuring protein levels in test cells are known inthe art. Many such methods employ antibodies (e.g., monoclonal orpolyclonal antibodies) that bind specifically to the HIN-1 polypeptide.In such assays, the antibody itself or a secondary antibody that bindsto it can be detectably labeled. Alternatively, the antibody can beconjugated with biotin, and detectably labeled avidin (a polypeptidethat binds to biotin) can be used to detect the presence of thebiotinylated antibody. Combinations of these approaches (including“multi-layer sandwich” assays) familiar to those in the art can be usedto enhance the sensitivity of the methodologies. Some of theseprotein-measuring assays (e.g., ELISA or Western blot) can be applied tolysates of test cells and others (e.g., immunohistological methods orfluorescence flow cytometry) to histological sections or unlysed cellsuspensions. Methods of measuring the amount of label will be depend onthe nature of the label and are known in the art. Appropriate labelsinclude, without limitation, radionuclides (e.g., ¹²⁵I, ¹³¹I, ³²S, ³H,or ³²P), enzymes (e.g., alkaline phosphatase or horseradish peroxidase),fluorescent moieties (e.g., fluorescein, rhodamine, or phycoerythrin),or luminescent moieties (e.g., Qdot™ nanoparticles supplied by theQuantum Dot Corporation, Palo Alto, Calif.). Other applicable assaysinclude quantitative immunoprecipitation or complement fixation assays.

[0119] Generally, the level of HIN-1 mRNA or protein in cancer cellswill be at least two-fold (e.g., three-fold, four-fold, five-fold,six-fold, seven-fold, eight-fold, 10-fold, 15-fold, 20-fold, 40-fold,60-fold, 80-fold, 100-fold, 500-fold, 1,000-fold, or higher-fold) lessthan in the normal cell counterpart of the cancer cell.

[0120] Methods of measuring the number of methylated C residues in theCpG sequences within the CpG island of the HIN-1 promoter are known inthe art. One such methodology is described in Example 4. In FIG. 8 isshown the nucleotide sequence (SEQ ID NO:19) of DNA that, in thehHIN-gene, lies immediately 5′ of the ATG initiation codon of the hHIN-1coding region and that includes the CpG island referred to above. Whilethe residue designated “N” in SEQ ID NO19 (FIG. 8) has not beenidentified, it is either a “C” or a “G”. All or part of the SEQ ID NO:19can be used in these assays. Furthermore, C residues within CpGsequences at the 5′ end of the HIN-1 coding region can be included insuch assays.

[0121] Standardizing such methylation assays to discriminate betweencancer and non-cancer cells of interest would involve experimentationfamiliar to those in the art. For example, the methylation status of theHIN-1 promoter region in DNA from sample cancer cells of interestobtained from a large number of patients can be compared to themethylation status of the HIN-1 promoter region in DNA from normal cellscorresponding to the cancer cells obtained either from the same patientsor from normal individuals. From such experimentation it will bepossible to establish a range of “cancer levels” of methylation and arange of “normal levels” of methylation. Alternatively, the methylationstatus of the HIN-1 promoter region in DNA from cancer cells of eachpatient can be compared to the methylation status of the HIN-1 promoterregion in DNA from normal cells (corresponding to the cancer cells)obtained from the same patient. In such assays, it is possible thatmethylation of as few as one cytosine residue could discriminate betweencancer and non-cancer cells.

[0122] Other methods for quantitating methylation of DNA are known inthe art. Such methods are based on: (a) the inability ofmethylation-sensitive restriction enzymes to cleave sequences thatcontain one or more methylated CpG sites [Issa et al. (1994) Nat. Genet.7:536-540; Singer-Sam et al. (1990) Mol. Cell. Biol. 10:4987-4989; Razinet al. (1991) Microbiol. Rev. 55:451-458; Stoger et al. (1993) Cell73:61-71]; and (b) the ability of bisulfite to convert cytosine touracil and the lack of this ability of bisulfite on methylated cytosine[Frommer et al. (1992) Proc. Natl. Acad. Sci. USA 89:1827-1831; Myöhänenet al. (1994) DNA Sequence 5:1-8; Herman et al. (1996) Proc. Natl. Acad.Sci. USA 93:9821-9826; Gonzalgo et al. (1997) Nucleic Acids Res.25:2529-2531; Sadri et al. (1996) Nucleic Acids Res. 24:5058-5059; Xionget al. (1997) Nucleic Acids Res. 25:2532-2534].

[0123] The following examples are meant to illustrate, not limit, theinvention.

EXAMPLES Example 1 Materials and Methods

[0124] Purification of Luminal Mammary Epithelial Cells.

[0125] The presence of normal and DCIS mammary epithelium in breasttissue samples was confirmed by examination of hematoxylin-eosin stainedfrozen sections. Breast tissue was minced into small pieces and digestedin DMEM/F12 medium (Life Technologies, Rockville, Md.) supplemented with1% fetal bovine serum (FBS) and 2 mg/ml collagenase I (Sigma; CatalogNo. C0130) and 2 mg/ml hyaluronidase (Sigma; Catalog No. H3506) at 37°C. for 2 hours with constant agitation. Cells were collected bycentrifugation (3,000 rpm for 10 min.), washed in 10 ml of PBS(phosphate buffered saline, Life Technologies), centrifuged again, andtreated with trypsin (5 ml 0.05% of Trypsin-EDTA; Life Technologies) at37° C. for 5 min. Cells were collected by centrifugation and resuspendedin 200 μl of PBE (PBS, 1% bovine serum albumin, 2 mM EDTA). Cells werepurified using anti-BerEP4 antibody coated magnetic beads (Dynabeads™;10 μl/˜1 million cells) (Epithelial Enrich; Dynal, Oslo, Norway). Theantibody coated magnetic beads were added to 200 μl of the cellsuspension containing approximately 10 cells. The mixture was incubatedon ice for 10-30 min. The beads with appropriate cells bound werepelleted by placing the bottom of the tube containing the mixture on amagnet. PBE containing cells not bound to beads was removed and thebeads were resuspended in 200 μl PBE. This washing procedure wasperformed 3 times. After the last wash, cells bound to the beads(Dynabeads™) were lysed in RNA lysis buffer (mRNA Direct Kit, Dynal) andRNA was isolated according to the manufacturer of the mRNA Direct KitmRNA isolation kit (Dynal).

[0126] Generation and Analysis of SAGE Libraries.

[0127] SAGE libraries were generated following a modified micro-SAGEprotocol, but including a 1% sodium dodecyl sulfate (SDS)washing/heating step following each enzymatic reaction in order toensure complete inactivation of the enzymes. [Velculescu et al. (1995)Science 270, 484-487; Polyak et al. (1997) Nature 389, 300-305; Lal etal. (1999) Cancer Res. 59, 5403-5607; Velculescu et al. (1999) NatureGenetics 23, 387-388].

[0128] Cell Lines and Tissue.

[0129] BT-20, BT-474, BT-549, Hs578T, MCF-7, MDA-MB-231, MDA-MB-468,MDA-MB-435S, SK-BR-3, T47D, UACC-812, UACC-893, and ZR-75-1 breastcancer cell lines were obtained from the American Type CultureCollection (ATCC; Manassas, Va.). SUM-44, SUM-52, SUM-102, SUM149,SUM-159, SUM-185, SUM-190, SUM-225, SUM-229, and SUM-1315 cell lineswere a generous gift of Dr. Steve Ethier (University of Michigan MedicalCenter). The HCC1937 BRCA1 minus cell line was obtained from Dr. GailTomlinson (University of Texas Southwestern Medical Center). The 21MT1,21MT2, 21NT, and 21PT cell lines were obtained from Dr. Arthur Pardee(Dana-Farber Cancer Institute). Cells were grown in media recommended bythe ATCC or by the establishing investigators. However, 48 hours priorto RNA extraction, all cell lines were switched to DMEM/F12 mediumsupplemented with 5% FBS in order to minimize gene expressiondifferences due to culture conditions. To test the effect of methyltransferase inhibitors, cells were grown in the presence of 25 μM5-aza-2′-deoxycytidine for 10 days, then harvested for RNA preparation.Primary tumors were obtained from consecutive surgeries from the Brighamand Women's Hospital and Massachusetts General Hospital, snap frozen ondry ice, and stored at −80° C. until use. Primary mammary epithelialcell cultures were initiated with cells purified from organoids asdescribed above for the purification of luminal mammary epithelial cellsand the cells were grown in MBEM medium (Clonetics, Walkersville, Md.).Laser capture microdissection (LCM) of frozen tumors was performedessentially as described. [Sgroi et al. (1999) Cancer Res.59:5656-5661].

[0130] Primary cultures of human bronchial epithelial cells werepurchased from Clonetics (Walkersville, Md.) and were cultured in thepresence or absence of 1 mM all-trans retinoic acid essentially aspreviously described [Koo et al. (1999) Am. J. Respir. Cell Mol. Biol.20:43-52].

[0131] Mouse organs and embryos were collected following protocolsapproved by the Animal Care and Use Committee of the Dana-Farber CancerInstitute.

[0132] RNA Preparation and Northern Blot Analysis.

[0133] RNA isolation, RT-PCR and Northern blot analysis were performedessentially as described [Polyak et al. (1997)]. Human multiple tissuenorthern blots and expression arrays were purchased from Clontech (PaloAlto, Calif.) and hybridized with a PCR-derived, full-length HIN-1 cDNAprobe according to the manufacturer's instructions.

[0134] mRNA in situ Hybridization.

[0135] Digitonin-labeled mouse HIN-1 riboprobes were generated and mRNAin situ hybridization was performed as described by Qian et al. [(2001)Genes Dev. 15:2533-2545].

[0136] hHIN-1 Methylation, Loss of Heterozygocity and Mutation Analysis.

[0137] Searching the draft of the human genome sequence with the hHIN-1cDNA sequence identified a genomic clone (CTB-36B8) containing theentire hHIN-1 gene, the proximal promoter region of which contained anapparent CpG island. To determine the location of methylated cytosines,genomic DNA was extracted from the cells, bisulfite treated and purifiedas previously described [Herman et al. (1998) Proc. Natl. Acad. Sci. USA95:6870-6875]. PCR amplification was performed using the followingprimers designed to amplify the coding strand (nucleotides −345 to +72)of bisulfite treated DNA: forward primer-5′-gagggaaagttttttttatttgg-3′(SEQ ID NO:9) and reverse primer-5′-caaaactaacaaaacaaaacca-3′ (SEQ IDNO:10). PCR reactions were performed in 50 μl reactions containing 16.6mM (NH₄)₂SO₄, 67 mM Tris pH 8.8, 6.7 mM MgCl₂, 10 mM β-mercaptoethanol,1.5 mM each deoxynucleotide, 35 ng of each primer, 1 μl of Platinum® Taq(Life Technologies) and 2 μl of bisulfite treated genomic DNA astemplate. Amplifications were performed using a “touch-down” protocol:initial denaturation 95° C. 3 min., 5 cycles of 95° C. 30 sec., 61° C. 1min, 70° C. 1 min.; 35 cycles of 95° C. 30 sec., 57° C. 1 min, 70° C. 1min.; followed by 70° C. 5 min. PCR products were subcloned into thepZERO1.0™ plasmid (Invitrogen) and 4-6 independent colonies weresequenced for each PCR product. Based on the sequence ofmethylated/unmethylated templates, PCR primers were designed for thespecific amplification of methylated or unmethylated DNA. After severaltests, the following primers were found to be highly specific and usedin all subsequent experiments: methylated DNA forward primers F1 (nt−209 to −186): 5′-gttaagaggaagttttcgaggttc-3′ (SEQ ID NO:11), F2 (nt−172 to −149): 5′-ggtacgggttttttacggttcgtc-3′ (SEQ ID NO:12), reverseprimer R2 (nt −37 to −58): 5′-aacttcttatacccgatcctcg-3′ (SEQ ID NO:13);unmethylated DNA forward primers F1 (nt −209 to −186):5′-gttaagaggaagtttttgaggttt-3′ (SEQ ID NO:14), F2 (nt −172 to −149):5′-ggtatgggttttttatggtttgtt-3′ (SEQ ID NO:15), reverse primer R2 (nt −37to −58): 5′-caaaacttcttatacccaatcctca-3′ (SEQ ID NO:16). PCRamplifications were performed as described above; PCR fragments wereanalyzed on 3% agarose gels.

[0138] For loss of heterozygocity studies, PCR forward(5′-tttccctgcttccacactagc-3′) (SEQ ID NO:17) and reverse(5′-agattaagaaggaattgacct-3′) (SEQ ID NO:18) primers were designed toamplify a CA repeat present in the CTB-36B8 genomic clone containing thehHIN-1 gene. PCR amplifications using ³²P end-labeled primers wereperformed essentially as described [Thiagalingam et al. (1996) Nat.Genet. 13(3):343-346]. Mutation screen was performed either onPCR-derived, full-length cDNA fragments or PCR fragments of individualexons amplified from genomic DNA using intron specific primers.

[0139] Generation of Recombinant hHIN-1 Protein and PolyclonalAnti-HIN-1 Antibodies.

[0140] Human HIN-1 encoding cDNA without the start methionine codon wasPCR amplified and subcloned into the pQE-30 expression vector(QIAexpress® Protein Purification System; Qiagen, Valencia, Calif.) inframe with an N-terminal hexahistidine tag and transformed intoMJ15[pREP4] bacteria. For large scale protein purification, a singlebacterial colony was inoculated into 20 ml of TB (Terrific Broth; LifeTechnologies) medium containing 200 μg/ml ampicillin and 25 μg/mlkanamycin, grown overnight at 37° C., and transferred into 1000 ml ofthe same medium the following morning. Once the OD₆₀₀ of the culture hadreached 0.6-0.8, protein expression was induced at by the addition ofisopropyl-β-D-thiogalactopyranoside (IPTG; 1 mM final concentration) tothe medium followed by incubation for an additional four hours. Bacteriawere collected by centrifugation and lysed by sonication in 50 ml oflysis buffer containing 8 M urea, 50 mM Tris (pH 7.7), 20 mM imidazole,1 M NaCl, 0.1% Triton X-100, 10 mM β-mercaptoethanol, and 20% ethanol.Cleared cell lysates were then incubated with 0.5 ml of Ni-NTA Agarose(Qiagen) for one hour at room temperature, followed by repeated washeswith lysis buffer. Bound proteins were then eluted in a buffercontaining 300 mM imidazole, 0.5 M NaCl, 50 mM Tris (pH), and 10%glycerol and dialyzed into PBS containing 10% glycerol at 4° C. prior touse.

[0141] Rabbit polyclonal antibody specific for hHIN-1 protein wasprepared by Zymed (South San Francisco, Calif.).

[0142] Generation of hHIN-1 Mammalian Expression Constructs andRecombinant Adenoviruses.

[0143] For stable constitutive expression in mammalian cells, hHIN-1encoding cDNA (or p53 encoding cDNA) was PCR amplified and subclonedinto the pCEP4 vector (Invitrogen, Carlsbad, Calif.). For the generationof a recombinant adenovirus the hHIN-1 cDNA was PCR amplified andsubcloned into the Kpn I-Xho I site of the pAd-Track-CMV adenoviralvector followed by recombination and adenovirus generation using theAd-Easy™ (Quantum Biotechnologies, Montreal, Canada) system [He et al.(1998) Proc. Natl. Acad. Sci. USA 95:2509-2514]. Expression of hHIN-1protein was confirmed by western blot analysis using anti-hHIN-1polyclonal antibodies.

[0144] Colony Assays, Cell Cycle Analysis and Western Blot Analysis.

[0145] For colony assay experiments, cells in 100 mm plates weretransfected with a mix of 42 μl of FuGene6 (Roche) and 21 μg of theindicated plasmid. 24 hours after transfection, 84% of the cells wereplated into 2 T25 tissue culture flasks in medium without a selectiondrug. Twenty-four hours later the medium was replaced with selectionmedium containing hygromycin. Colonies were allowed to grow for 2 weeksafter which they were visualized by crystal violet staining.

[0146] For cell cycle analyses, cells were plated into 6 well tissueculture plates (1×10⁵ cells/well) and infected with 2 μl (˜50-100 m.o.i.(multiplicity of infection)) of Ad-Track-GFP (Green FluorescenceProtein) or Ad-Track-Histag-HIN-1 replication defective recombinantadenoviruses. For some of these experiments cells were grown in mediumcontaining 0.2% serum in order to maximize a potential growth inhibitoryeffect.

[0147] For western blot analysis, cells and media from 293 cellstransfected with pCEP4 (control vector containing no expressible cDNAinsert), pCEP4-Histag-HIN-1 (vector containing cDNA encoding hHIN-1fused to hexahistidine) constructs, and MCF10A or SUM159 cells infectedwith Ad-Track-GFP or Ad-Track-Histag-HIN-1 were lysed in denaturing ureabuffer (as described for purification of recombinant hHIN-1 frombacteria) and the lysates were incubated with Ni-NTA beads. Proteinsbound to the beads were eluted off the beads, subjected to SDS-PAGE, andimmunoblotted with rabbit polyclonal antibody specific for hHIN-1diluted 1: 1,000.

Example 2 Generation and Analysis of SAGE Libraries

[0148] SAGE libraries were generated from two independent cases of DCISand two samples of luminal mammary epithelium and analyzed. One of theDCIS tumors was a high grade, comedo DCIS (DCIS1), while the other onewas a solid, intermediate-grade DCIS (DCIS2). Normal luminal mammaryepithelial cells were derived from corresponding contralateralprophylactic mastectomy tissue (Normal 2) obtained from the DCIS2 caseor from breast reduction surgery on an unrelated patient (Normal 1).Luminal mammary epithelial cells and breast cancer cells were purifiedusing anti-Ber-EP4 coated magnetic beads, and SAGE libraries weregenerated using a modified version of a micro-SAGE protocol.

[0149] From the four SAGE libraries 160,046 tags were obtained,approximately 40,000 from each library. With this high number of tags,it was possible to compare the expression levels of close to 30,000unique transcripts. Pair-wise comparison of these SAGE librariesidentified several differentially expressed tags. 97 tags were elevatedat least 10-fold in one or the other DCIS library, while 132 tags wereat least 10-fold more abundant in the normal libraries. Interestinglythere was only 1 tag that was highly elevated (about 113 to 95-fold) andthere were 9 tags that were 10-fold decreased in both DCIS libraries.These 10 tags were searched against ˜85 other SAGE libraries derivedfrom a variety of normal and cancerous tissue types. One of the tags wasparticularly interesting, since it was present only in the two normalluminal mammary epithelial cell SAGE libraries. This finding indicatedthat the tag was derived from a transcript from which a polypeptideassociated with mammary epithelium specific function is translated.Database searches identified a 461 bp human full-length Unigene cDNAclone corresponding to this tag. The full length cDNA (461 bp) ispredicted to include a coding sequence (SEQ ID NO:3) (FIG. 1A) encodinga small protein (the hHIN-1 polypeptide) of 104 amino acids (˜11 kDa)(SEQ ID NO:1) (FIG. 2A). This conclusion was confirmed by in vitrotranscription/translation experiments. The protein was designated humanHIN-1 (hHIN-1). The hHIN-1 protein contains a putative signal peptide(see above), and is predicted to be secreted. This consideration and thefact that the gene product regulates cell proliferation (see below)indicates that hHIN-1 is a novel cytokine. A database search (dbesttblastx) also identified a homologous mouse cDNA sequence (SEQ ID NO:7)(FIG. 3A) encoding a polypeptide (mHIN-1; SEQ ID NO:5) (FIG. 4A) of thesame length as the hHIN-1 polypeptide and with an analogous signalpeptide. Due to its homology to hHIN-1 (60.8% identity at the amino acidlevel), it is likely that mHIN-1 has essentially the same function ashHIN-1. The same database search revealed a rat partial cDNA sequence(SEQ ID NO:20) (FIG. 9A) encoding a polypeptide (SEQ ID NO:21) highlyhomologous to both hHIN-1 and mHIN-1. The rat polypeptide is 62%identical to the hHIN-1 polypeptide and is 84% identical to the mHIN-1polypeptide. It seems likely that this amino acid sequence (SEQ IDNO:21) is rat HIN-1 (rHIN-1) missing several N-terminal amino acids andthat, like mHIN-1, rHIN-1 has the same function as hHIN-1. In FIG. 10 isshown the amino acid sequences of hHIN-1, mHIN-1, and the partialsequence of rHIN-1 aligned for maximum homology. Amino acids indicatedbelow the aligned sequences are those that are common to at least two ofthe polypeptides at the relevant positions.

Example 3 hHIN-1 Expression in Normal Tissues and in Breast Carcinomas

[0150] The lack of SAGE tags corresponding to the hHIN-1 mRNA in 85other SAGE libraries suggested an intriguing tissue specific pattern ofhHIN-1 expression. To confirm this cell type-specific expressionpattern, ³²P-labeled hHIN-1 cDNA was hybridized to an expression arraypanel consisting of dots of mRNA from 76 human adult and fetal tissuetypes bound to a blotting membrane. In addition to mammary gland, thehHIN-1 gene appears to be highly expressed in lung, trachea, salivarygland, prostate, esophagus, duodenum, fetal lung and fetal kidney (FIG.5A). Lower levels of hHIN-1 mRNA expression were detected in pancreas,pituitary gland, lymph node and accumbens nucleus. To verify theidentity of the signal detected on the dot blots, multiple tissuenorthern blots were also exposed to ³²P-labeled HIN-1 cDNA. The dataconfirmed that the hybridizing RNA corresponds to a single hHIN-1 mRNA(FIG. 5B). The high expression of hHIN-1 in tissues that containepithelia-producing mesenchymal tissue suggest that HIN-1 might play arole in epithelial branching morphogenesis.

[0151] SAGE analysis indicated hHIN-1 expression levels at least 20-foldlower in DCIS tissue than in two different normal luminal mammaryepithelia. In situ hybridization was performed to confirm hHIN-1expression at the cellular level. hHIN-1 is highly and specificallyexpressed in normal luminal epithelial cells of small ducts and lobules,but not that of large ducts (FIGS. 5C and 5D). In contrast, nohybridization signal was detected in DCIS (FIG. 5E). Northern blotanalysis was performed to further evaluate hHIN-1 expression levels inmultiple independent normal breast organoids, in primary mammaryepithelial cell cultures and in breast cancer cell lines. Arepresentative result of these experiments is shown in FIG. 5F. Highlevels of hHIN-1 expression were detected in freshly isolated breastorganoids, but not in cultured normal mammary epithelial cells nor inmost breast cancer cell lines tested by northern blot. Furthermore, wewere unable to detect hHIN-1 mRNA by RT-PCR in 89% (25/28) of breastcancer cell lines. hHIN-1 expression was dramatically up-regulated inpregnant epithelium. The expression of hHIN-1 was further investigatedby real-time PCR analysis of LCM dissected primary tumors. The resultsfrom 33 representative cases are shown in FIG. 5G. “Fold difference”indicates the ratio of hHIN-1 mRNA levels in normal versus cancerousepithelium isolated from the same patient. Only 4 tumors were found toexpress detectable hHIN-1 mRNA, while the majority of tumors (78%) badno detectable hHIN-1 expression. These primary tumors included in situ,invasive ductal and lobular carcinomas, and hHIN-1 expression was lostregardless of tumor stage and histological type. Thus, loss of hHIN-1expression is an early and frequent event in human breast carcinomas.Interestingly, northern blot analysis of 40 primary lung tumors alsorevealed dramatically reduced (>90% compared to normal lung tissue)hHIN-1 mRNA levels.

Example 4 hHIN-1 Expression is Silenced by Methylation

[0152] The loss of hHIN-1 expression in the majority of breast cancerssuggested a tumor suppressor function for hHIN-1. In order to evaluateif HHIN-1, following the Knudson model, undergoes biallelic inactivationin breast cancers, we performed LOH (loss of heterozygocity) andmutational analyses of the HIN-1 gene. A search of a draft of the humangenome sequence using the sequence of hHIN-1 cDNA resulted in theidentification of a genomic clone containing the entire hHIN-1 gene andan adjacent polymorphic CA repeat as being suitable for LOH analysis.Analysis of this CA repeat in 43 primary tumors revealed LOH in 25% ofthe informative cases. However, sequencing the other allele revealed nomutations. Similarly, PCR analysis of breast cancer cell lines detectedno homozygous deletions, and sequence analysis of the hHIN-1 cDNA infour cell lines that expressed hHIN-1 revealed no mutations. Therefore,the loss of hHIN-1 expression in breast cancer is unlikely to be due togenetic events. It seemed likely that epigenetic mechanisms (such as DNAmethylation) might instead be responsible. This hypothesis wasstrengthened by the presence of a CpG island in the proximal promoterregion of the hHIN-1 gene.

[0153] To investigate the potential role of DNA hypermethylation insilencing hHIN-1 expression, the sequence of its promoter region inbisulfite treated DNA isolated from normal mammary epithelial cells andhuman breast cancer cell lines was analyzed. FIG. 6A shows the frequencyof methylation of up to 56 CpG sites in the hHIN-1 promoter regions in4-10 individual PCR product clones derived from each cell line ortissue. Also shown in FIG. 6A are the relative levels of hHIN-1 mRNA inthe relevant cells. In DNA from some cells, shorter regions (“−304 to+31 ” or “−532 to −281 ”) were analyzed while in others a longer region(“−532 to +31”) was analyzed. The “−532 to +31” region contained thesequence shown in FIG. 8 (SEQ ID NO:19) and the first 12 nucleotides ofthe hHIN-1 coding sequence. The “−304 to +31” region contained the 3′323 nucleotides of SEQ ID NO:19 and the first 12 nucleotides of thehHIN-1 coding sequence. As shown in FIG. 6A, virtually all of the CpGsin the proximal promoter region analyzed are methylated in breast cancercells with no hHIN-1 expression (ZR-75-1, T47D, and BT474), whileessentially no methylated CpGs were found in normal mammary epithelialcell samples (“Normal”). The data shown for “Normal” were pooled fromdata obtained from separate analyses of DNA from each of threeindependent normal mammary epithelial cell samples. Normal breast tissuesamples from an 18-year old and 34-year old patient (“Normal (18 yo)”and “Normal (34 yo)”, respectively) were also analyzed. The analysis ofthe tissue from the 34 year old patient indicated a high level of hHIN-1mRNA in the tissue and a low level of methylation of the hHIN-1 promoterregion. The analysis of the tissue from the 18-year old patient alsoindicated a high level of hHIN-1 mRNA in the tissue but with significantmethylation of at least the distal end of the hHIN-1 promoter region. Itis possible that hHIN-1 gene expression is affected more potently bymethylation of CpG sites more proximal to the transcription initiationsite than by methylation of CpG sites more distal to the transcriptioninitiation site. Alternatively, or in addition, there may beage-dependent differences in methylation of the HIN-1 gene; suchage-dependent differences in methylation have been observed in othergenes. Interestingly, some breast cancer cell lines (BT-549, SK-BR-3,SUM159 and SUM149) and parts of one tumor (T44) had some expression ofhHIN-1 mRNA, although at much lower levels than that of normal cells(and detectable by RT-PCR but not by Northern blot analysis), and theirpromoter regions were found to be partially methylated (FIG. 6B). TheSUM225 breast cancer cell line initially had hHIN-1 mRNA levelscomparable to that of normal mammary epithelium (FIG. 5C); however, itprogressively lost hHIN-1 expression in later passages (FIG. 6B) and itspromoter region became highly methylated (FIG. 6A). In a pancreaticcancer cell line (ASCP) no hHIN-1 mRNA was detected and a high degree ofmethylation of the hHIN-1 promoter region was observed. In addition, ahigh degree of methylation of the hHIN-1 promoter region was found intwo prostate cancer cell lines (PC3 and LNCP). A moderate level of thehHIN-1 promoter methylation was detected in DNA from a pool of four lungcarcinoma tissue samples (“Lung CA”). Thus, there appears to be a strongcorrelation between hHIN-1 promoter region methylation status and mRNAlevels in all of these breast cancer cell lines examined.

[0154] To test for the consequence of promoter methylation on hHIN-1expression, the effect of a DNA methyltransferase inhibitor(5-aza-2′-deoxycytidine; “5aza-C”) on hHIN-1 mRNA levels was analyzed.Six breast cancer cell lines with no detectable expression of hHIN-1were grown in the presence or absence of 25 μM 5aza-C for three to tendays, then lysed for RNA and DNA preparation. The expression of hHIN-1mRNA was determined by RT-PCR analysis, while the extent of promotermethylation was evaluated by sequence analysis of bisulfite treatedgenomic DNA. As shown in FIG. 6A, 5aza-C treatment lead to there-expression of hHIN-1 mRNA in all six cell lines. This hHIN-1re-expression correlated with a decrease in the extent of promotermethylation (FIG. 6A; compare the degree of hHIN-1 promoter regionmethylation in ZR-75-1 cells not treated with 5aza-C (“ZR-75-l”) andZR-75-1 cells treated with 5aza-C (“ZR-75-1-AC”)). Based on these data,it is concluded that methylation is at least partially responsible forthe loss of hHIN-1 expression in breast cancer cell lines.

[0155] A methylation specific PCR (MSP) assay was developed in order toanalyze the methylation status of the hHIN-1 promoter region in primarybreast tumors. Primers were designed to amplify methylated orun-methylated DNA following bisulfite treatment. Using this approach,the HIN-1 promoter regions of three independent normal breast tissueswere found to be completely unmethylated, while that of a positivecontrol breast cancer cell line (ZR75-1) was completely methylated.Analysis of 28 cell lines and 101 primary tumors showed that the hHIN-1promoter regions of 89% of the cell lines and 74% of primary tumors werecompletely or partially methylated (representative examples are shown inFIG. 6C). Of these primary tumors, 31 (23 methylated and 8 unmethylated)were analyzed by real-time PCR. All of the methylated tumors and 6 ofthe unmethylated ones lacked hHIN-1 mRNA. These results indicate thataberrant hHIN-1 promoter region hypermethylation and subsequent lack ofexpression occur frequently in breast cancers. However, other mechanismsmight be responsible for silencing hHIN-1 in a small fraction of tumors.Whether hHIN-1 promoter region hypermethylation in the tumors correlateswith any patient or tumor characteristics was analyzed. The results aresummarized in Table 1. Among the parameters analyzed, only theassociation of lack of hHIN-1 promoter region hypermethylation with highhistologic grade appeared to be statistically significant. The lack ofhHIN-1 promoter region methylation coupled with the lack of hHIN-1expression observed in these high-grade tumors indicates that in thehigh histologic grade tumors either (1) hHIN-1 expression is silenced bysome mechanism other than promoter region hypermethylation, e.g., lossof a crucial transcription factor or that (2) there is a deficiency in adownstream mediator of the hHIN-1 signaling pathway. Since high-gradetumors in general have worse overall prognosis, HHIN-1 promoter regionmethylation status may predict the clinical behavior of tumors. TABLE 1hHIN-1 promoter region methylation status in breast cancers with variouscharacteristics and from patients of different ages. Yes No HIN-1 NumberNumber Total Methylation (% of total) (% of total) Number Patients 73(72) 28 (28) 101  Age, y ≧40 60 (70) 26 (30) 86 ≦40 12 (86)  2 (14) 14≧50 33 (67) 16 (33) 49 ≦50 39 (76) 12 (24) 51 Tumors Estrogen receptor(ER) positive 43 (78) 12 (22) 55 negative 26 (67) 13 (33) 39Progesterone receptor (PR) positive 39 (80) 10 (20) 49 negative 30 (67)15 (33) 45 ER/PR positive 34 (79)  9 (21) 43 negative 21 (64) 12 (36) 33Her2/neu/erbB2 positive 38 (70) 16 (30) 54 negative 27 (77)  8 (23) 35Histology grade high 26 (60) 17 (40) 43 low/intermediate 35 (80)  9 (20)44 Lymph node status positive 36 (78) 10 (22) 46 negative 31 (67) 15(33) 46

[0156] The results of two-sided X² tests for the occurrence of equalfrequencies of hHIN-1 promoter region hypermethylation in the indicatedsubgroups of the indicated patient and tumor groups were as follows: age(P=0.22), estrogen receptor expression (P=0.21), progesterone receptorexpression (P=0. 16), estrogen receptor expression and progesteronereceptor expression (“ER/PR”) (P=0.13), Her2/neu expression (P=0.48),histology grade (P=0.052), and lymph node status (P=0.35).

Example 5 hHIN-1 is a Novel Growth Inhibitory Cytokine

[0157] In order to investigate the effect of constitutive hHIN-1expression on mammary cell growth, a mammalian expression constructcontaining hHIN-1 polypeptide encoding cDNA (pCEP4-HIN-1) wastransfected into various breast cancer cell lines. Stable transfectantswere selected in the presence of hygromycin for 2 weeks. Cell colonieswere visualized by crystal violet staining. hHIN-1 expression led to adramatic decrease in colony numbers in BT549 cells and to a lesserdegree in MDA-MB-435 cells compared to control pCEP4 transfected cells.In contrast, p53 effectively inhibited cell growth in both cell lines.The different effects of hHIN-1 expression on cell growth in the twocell lines indicate that certain cell lines might be non-responsive tohHIN-1 due to some other defect in the hHIN-1 signaling pathway.

[0158] To confirm that hHIN-1 is a secreted protein, an immunoblotanalysis of cell extracts and media separated from cells transientlytransfected with a mammalian expression construct or infected with arecombinant adenovirus expressing a hexahistidine tagged hHIN-1 proteinwas performed (FIG. 7). Using a rabbit polyclonal anti-hHIN-1 antibody,a ˜11 kDa protein was detected in both cell lysates and media from cellsexpressing hHIN-1 but not in cell lysates or media from control hHIN-1non-expressing cells. The hHIN-1 protein migrates as a doublet onSDS/Tricine (the buffer use for the SDS-PAGE) gels.

Example 6 Expression of HIN-1 at Different Stages of Development

[0159] The UGRP-1 (Uteroglobin related protein-1) gene is related to theHIN-1 gene and is a downstream target of the Nku2.1 homeogene [Niimi etal. (2001) Ann. N. Y Acad. Sci. 923:43-58]. Based on amino acid sequenceand predicted structural homology, both proteins belong to thesecretoglobin family of small, secreted proteins [Singh et al. (2000)Ann. N.Y. Acad. Sci. 923:43-58]. Therefore, HIN-1 and UGRP-1 are nowalso called SCGB3A1 and SCGB3A2 acronyms for secretoglobin 3A1 and 3A2,respectively [Klug et al. (2000) Ann. N.Y. Acad. Sci. 923:348-354]. Thesilencing of hHIN-1 expression in human breast carcinomas and decreasedcolony growth of breast cancer cells following overexpression indicate atumor suppressor role for the HIN-1 gene.

[0160] Experiments described above showed that the expression of HIN-1in humans is restricted to organs composed of branching epithelia. Todetermine if the expression of UGRP-1 overlaps with that of HIN-1, theexpression of both genes was analyzed in various adult and developinghuman and mouse organs by northern blot hybridization (FIGS. 11A-C). Inadult organs, UGRP-1 exhibits lung- and trachea-specific expression inboth humans and mice (FIGS. 11A and B). While the highest level ofmHIN-1 expression is detected in the lung, a low level of expression wasalso detected in the heart, stomach, and small intestine of the mouse(FIG. 11B). During development of the mouse high mHIN-1 and UGRP-1expression was first detected in the lung at E17.5-E18.5 (day 17.5 today 18.5 of embryonic life) (FIG. 11C and FIGS. 12G and H). A low levelof mHIN-1 mRNA expression was detectable in embryos at E6.5 (day 6.5 ofembryonic life) (FIG. 11C); however, mRNA in situ hybridization revealedthat the source of the mHIN-1 mRNA was likely contaminating uterinetissue (FIGS. 12N and O).

[0161] Northern analysis showed no mHIN-1 and UGRP-1 expression in themouse mammary gland (data not shown). To test for a level of expressionbelow the detection sensitivity of northern analysis, the expression ofmHIN-1 and UGRP-1 in the mouse mammary gland at different developmentalstages was studied (FIGS. 11D and E). Consistent with the importance ofhHIN-1 in human breast carcinomas, expression of both mHIN-1 and UGRP-1mRNA was observed in the mouse mammary gland. Specifically, theexpression of UGRP-1 mRNA is up-regulated in early gestation and thenbecomes undetectable after day 10.5 p.c. (post coitum). In contrast,after initial up-regulation, mHIN-1 expression was maintained at a lowlevel throughout gestation and lactation, followed by a second increaseduring involution (FIGS. 11D and E). Since in normal human mammaryepithelial cells and in breast carcinomas the expression of hHIN-1correlated with that of the transcription factor C/EBPδ [Porter et al.(2001) Cancer Res. 61:5697-5702], the levels of C/EBPδ in multiple mousetissues were analyzed in order to determine if C/EBPδ could be anup-stream regulator of HIN-1 expression. Although C/EBPδ was detected inthe tissues at the developmental stages at which mHIN-1 was expressed,the levels of C/EBPδ mRNA did not show a strict correlation with thoseof mHIN-1 mRNA (FIGS. 11B-E).

[0162] Experiments described above showed that in the human mammarygland hHIN-1 expression is restricted to luminal mammary epithelialcells. To further analyze the expression of mHIN-1 at the cellular levelin the mouse, mRNA in situ hybridization of adult lung, trachea andmouse embryos at different stages of embryogenesis was performed (FIGS.12A-O and data not shown). mHIN-1 is highly and specifically expressedin the glandular epithelium lining the trachea and in bronchi both inadult mouse lung and in the lungs of embryonic mice at E17.5-E18.5(FIGS. 12A-L). Similar to UGRP-1, HIN-1 was also expressed in theepithelial cells of pregnant mouse uterine glands (FIGS. 12N and O).

[0163] The above pattern of mHIN-1 expression in adult and developingembryos strongly suggests a role for HIN-1 in terminal differentiationof epithelial cells. To test this hypothesis, the expression of hHIN-1mRNA by RT-PCR during retinoic acid-induced mucinous differentiation ofprimary human bronchial epithelial cells was analyzed (FIG. 13). Primaryhuman bronchial epithelial cells underwent squamous epithelialdifferentiation and expressed squamous cell markers such astransglutaminase I (Tgase I) in the absence of all-trans retinoic acid(FIG. 13). Following retinoic acid treatment in an air-interfaceculture, human bronchial epithelial cells differentiated into mucinouscells as demonstrated by the expression of genes specific for themucinous phenotype such as MUC2 (FIG. 13) [Koo et al. (1999) Am. JRespir. Cell Mol. Biol. 20:43-52]. In this in vitro differentiationsystem the expression of hHIN-1 mRNA correlated with the loss of asquamous marker (Tgase I) and preceded the induction of a marker formucinous differentiation (MUC2) following retinoic acid treatment. Suchexpression kinetics are consistent with a role for HIN-1 as an inducerof this process.

Example 7 Drosophila Homologues of HIN-1

[0164] Two previously uncharacterized Drosophila proteins (Drosophilagenes GC130681 and GC13674) were identified as showing limited (˜30%)homology to hHIN-1 and human UGRP-1 (FIG. 14A). The identification ofthese HIN-1/UGRP-1 homologues is particularly interesting, since to dateno secretoglobins have been identified in non-mammalian species. Theevolutionary relationship of these Drosophila proteins to members of thesecretoglobin family is depicted in FIG. 14B.

[0165] The amino acid sequence of the GC130681 protein is designated SEQID NO:28 and the nucleotide sequence of cDNA encoding the GC130681protein is designated SEQ ID NO:29 (FIG. 15). The amino acid sequence ofthe GC13674 protein is designated SEQ ID NO:30 and the nucleotidesequence of cDNA encoding the GC13674 protein is designated SEQ ID NO:31(FIG. 16). The amino acid sequence of human UGRP-1 is designated SEQ IDNO:32.

[0166] To determine if the function of HIN-1 is conserved betweenmammals and fruit flies, the expression of the GC130681 gene duringDrosophila development was analyzed by mRNA in situ hybridization.Expression of GC130681 mRNA was detected in the tracheal system of stage15-16 embryos. This finding correlates well with the above-describedstudies on mHIN-1 expression during mouse embryo development.

[0167] It should be understood that various modifications can be made tothe above-described embodiments without departing from the spirit of theinvention. Accordingly, the invention is limited only by the followingclaims.

What is claimed is:
 1. An isolated DNA comprising a nucleic acidsequence encoding a polypeptide consisting of SEQ ID NO:22.
 2. The DNAof claim 1, wherein the nucleic acid sequence encodes a polypeptideconsisting of SEQ ID NO:1.
 3. The DNA of claim 1, wherein the DNAcomprises SEQ ID NO:3.
 4. The DNA of claim 1, wherein the DNA comprisesof SEQ ID NO:23.
 5. A vector comprising (a) a nucleic acid sequence that(i) encodes a polypeptide that inhibits proliferation of breast cancercells, and (ii) hybridizes under highly stringent conditions to a probeconsisting of a sequence that is the complement of SEQ ID NO:3. (b) thecomplement of the nucleic acid sequence.
 6. The vector of claim 5,wherein the nucleic acid sequence is operably linked to atranscriptional regulatory element (TRE).
 7. A cell comprising thevector of claim
 5. 8. An isolated polypeptide comprising: (a) a proteinthat inhibits proliferation of breast cancer cells and is encoded by anucleic acid sequence that hybridizes under highly stringent conditionsto a probe consisting of a sequence that is the complement of SEQ IDNO:3; or (b) the protein, except for one or more conservative amino acidsubstitutions.
 9. The polypeptide of claim 8, wherein the polypeptidecomprises the amino acid sequence of SEQ ID NO:22.
 10. The polypeptideof claim 8, wherein the polypeptide comprises the amino acid sequence ofSEQ ID NO:1.
 11. A method of making a polypeptide, the method comprisingculturing the cell of claim 7 and extracting the polypeptide from theculture.
 12. A method of inhibiting proliferation of a cancer cell, themethod comprising contacting the cancer cell with the polypeptide ofclaim
 8. 13. The method of claim 12, wherein the contacting is in vitro.14. The method of claim 12, wherein the cancer cell is in a mammal. 15.The method of claim 12, wherein the cancer cell is a breast cancer cell.16. The method of claim 14, wherein the contacting comprisesadministering the polypeptide to the mammal.
 17. The method of claim 14,wherein the contacting comprises administering a polynucleotide encodingthe polypeptide to the mammal.
 18. The method of claim 14, the methodcomprising: a) providing a recombinant cell that is the progeny of acell obtained from the mammal and has been transfected or transformed exvivo with a nucleic acid encoding the polypeptide; and b) administeringthe cell to the mammal.
 19. A method of identifying a compound thatenhances inhibition of proliferation of cancer cells, the methodcomprising: a) providing a first and a second plurality of cancer cells;b) combining a test compound, the first plurality of cancer cells, andthe polypeptide of claim 8; c) combining the second plurality of cancercells and the polypeptide of claim 8; and d) determining the levelproliferation of the first plurality of cancer cells, wherein adecreased level of proliferation of the first plurality of cancer cells,as compared to the second plurality of cells, indicates that the testcompound enhances inhibition of proliferation of cancer cells by thepolypeptide.
 20. A method of diagnosis, the method comprising: (a)providing a test cell; and (b) measuring the level of expression of aHIN-1 gene in the cell, wherein lack of expression of the HIN-1 gene ora low level of expression of the HIN-1 gene is an indication that thetest cell is a cancer cell.
 21. The method of claim 20, whereinexpression of the HIN-1 gene is measured as a function of the level ofHIN-1 mRNA in the cell.
 22. The method of claim 20, wherein theexpression of the HIN-1 gene is measured as a function of the level ofHIN-1 polypeptide in the cell.
 23. A method of diagnosis, the methodcomprising: (a) providing a test cell; and (b) determining the degree ofmethylation of a HIN-1 promoter region in the test cell, wherein a highdegree of methylation of the HIN-1 promoter region is an indication thatthe test cell is a cancer cell.
 24. The method of claim 23, wherein thetest cell is a breast cell.
 25. An isolated polypeptide comprising (a) afunctional fragment of the polypeptide of claim 8; or (b) the functionalfragment, except for one or more conservative amino acid substitutions.26. An isolated DNA comprising a fragment of the nucleic acid with SEQID NO:3, wherein the fragment comprises nucleotides 55 and 56 of SEQ IDNO:3.
 27. An antibody that binds to the polypeptide of claim
 8. 28. Theantibody of claim 27, wherein the antibody is a monoclonal antibody. 29.The antibody of claim 27, wherein the antibody is a polyclonal antibody.30. A method of treatment comprising identifying a patient as havingcancer cells in which (a) HIN-1 gene expression is low or (b) a HIN-1promoter region is methylated; and treating the patient with a compoundthat reduces methylation of the HIN-1 promoter region.
 31. A method ofidentifying a compound that replaces the function of HIN-1 in cells thatdo not express HIN-1, the method comprising: (a) providing a first cellthat does not express HIN-1; (b) providing a second cell that doesexpress HIN-1; (c) treating the first cell and the second cell with atest compound; and (d) determining whether the test compound decreasesproliferation of the first or the second cell, wherein a compound thatdecreases proliferation of the first cell but not the second cell canpotentially replace the function of HIN-1 in cells that do not expressHIN-1.
 32. A method of treatment comprising identifying a patient ashaving cancer cells in which (a) HIN-1 gene expression is low or (b) aHIN-1 promoter region is methylated; and treating the patient with acompound that induces expression of a gene with a methylated promoterregion
 33. The method of claim 23, wherein the cell is a pancreaticcell.
 34. The method of claim 23, wherein the cell is a prostate cell.